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An approach to AIDS therapy using hyperthermia and membrane modification
Medical Hypotheses (1988) 27. 163-165 fQ Longman Group UK Ltd 1988
An Approach Hyperthermia MILTON Unwersity
to AIDS Therapy Using and Membrane Modification
B. YATVIN of Wisconsin
Medical
School,
420 N. Charter St. Madison,
WI 53706, USA.
the biophysical characteristics of cell membranes by diet and Abstract - Altering membrane perturbing agents markedly influences thermosensitivity of cells. Likewise, manipulation of viral envelopes either by altering their lipid composition by diet or by the use of agents that perturb the lipid envelope influence infectivity of enveloped viruses and the progression of viral disease. The use of hypet-thermia and envelope modification as a combined approach to treat AIDS has until now neither been suggested nor attempted. On the basis of my previous work and a review of the literature, I theorize that the combination of hyperthermia with procedures designed to alter the viral envelope will likely result in an increased viral sensitivity and be useful clinically for treatment of patients with enveloped viral diseases such as AIDS.
Introduction Over the years. my studies have led me to conclude that the initial effect of hyperthermia on cells is mediated via the heat-induced disorganization of membrane lipids. This disordering effect can be further enhanced by modifying the membrane either by diet or membrane active agents (1). I have postulated that such lipid disorganization modifies the membrane’s biophysical properties and leads to passive changes in transmembrane permeability, shifts in surface charge, and altered stereoorganization of macromolecules within the membrane (l-9). In support of the above, studies in our laboratory have shown that dietary alteration of the lipids of cell membranes has an effect on cellular thermosensitivity. and that membrane active agents
have a similar effect on the sensitivity of cells to heat (l-9). Consistent with this view are reports by others showing that treatment of mammalian cells with anaesthetics (10). alcohol (11) or highly unsaturated fatty acid diets (12) increases cell killing by hyperthermia both in vivo and in vitro. From the foregoing. it is likely that cell membranes play a critical role in determining the thermal sensitivity of cells. On the basis of the above, I propose that more effective treatment of enveloped viral disease will result when therapy includes membrane modification in conjunction with hyperthermia. Discussion
The membrane of enveloped viruses can be modified in viwo by removing cholesterol (13) or
163
164 by modifying the diet and growth conditions of the host cell both in vitro and in viuo (14, 15). When fluidity of the viral membrane was increased by removing cholesterol, infectivity of the vesicular stomatitis virus was reduced (13). Furthermore, the ordered state of membrane lipids can markedly affect protein interaction between and within membranes, including the presentation and exposure of proteins from the membrane (16) and the lateral movement of proteins within the membrane (17). Such changes in organization of cell and viral membranes may, in turn, mediate viral etiology, particularly the stages of attachment, penetration, assembly and release. For example, if viral or host cell membrane proteins are not sufficiently exposed (16), viral attachment will be inhibited. Likewise, the lateral mobility of proteins in a cell membrane (17) may affect the entry of envelope proteins into the host cell membrane and influence the assembly of the virus envelope. Viral assembly, budding and release reportedly are affected both by a low NaCI environment and by the ratio of cholesterol to phospholipid in the plasma membrane of the host cell (18). A glycoprotein involved in the maturation of a temperature-sensitive vesicular stomatitis virus did not become incorporated into the virus at a temperature of 40” C (19). At this non-permissive temperature, an appreciable amount of the viral glycoprotein reaches the plasma membrane with some being released into the medium. However, until the temperature was lowered, the viral glycoprotein remained on the cell surface and virions were not formed (19). Thus, it appears that virion assembly requires a less fluid host membrane. The above results indicate that host cell membrane organization has a major influence on the life cycle of viruses. In addition, the special lipid compound AL721 which fluidizes membranes 916), although not curative, beneficially alters the etiology of AIDS (Dr. M. Shinitzky, pers. comm.). Furthermore, there are many small hydrophobic compounds which fluidize lipid bilayers (20), some of which have already been used as antiviral agents (21-30). Of particular interest are the hydrophobic lipid perturbing molecules adamantane and butylated hydroxytoluene (BHT) and their related derivatives which have antiviral activity against enveloped viruses (21-30). The use of BHT has already been suggested as an approach to AIDS therapy in combination with reverse
MEDICAL HYPOTHESES
transcriptase inhibitors and perhaps biological response modifiers (29). The incorporation of BHT into the viral envelope causes perturbation (23) and in the case of adamantane may disrupt the envelope (21). Furthermore, when BHT is incorporated into host cell membranes, it interferes with viral budding in chicken cells (29) and adamantane affects the assembly of bacteriophage PM2 (21). In the latter case, it has been suggested that the cell membrane can function and grow with lipid alkyl chains in either the “ordered” or “disordered” state, but that the “ordered” state must be maintained for PM2 assembly to occur (21). BHT appears to have only a minimal effect on host cell membranes (30) but pretreatment of chicken embryo cells was found to inhibit the synthesis of Newcastle disease virus progeny by about 65% (25). BHT, which effectively fluidizes cell membranes of the V-,9 Chinese hamster lung fibroblasts, did not sensitize these cells to hyperthermic insult at 44.5” C (31). The above reports are consistent with idea that viral activity will be inhibited to a greater extent by membrane modification and heat that host cells. Conclusion I suggest the following as a logical approach for treating enveloped viral diseases such as AIDS: the use of hyperthermia with anaesthetics, dietary modification, or small hydrophobic compounds such as BHT and adamantane, either alone or in combination, tb maximally disrupt the viral envelope and alter the progression of viral disease. References 1. Yatvin. M B. The influence of lipid composition and procaine on hyperthermic death of cells. Int. .I. Radiat. Biol. 32: 513-521. 1977. 2. Yatvin M B, Clifton K H and Dennis W H. Hyperthermia and local anaesthetics: Potentiation of survival in tumor-bearing mice. Science 205: 195-196. 1979. 3. Hidveni E J. Yatvin M B. Dennis W H and Hidveei E. Effect” of altered membrane lipid composition -and killing of ascites cells. procaine on hyperthermic Oncology 37: 360-363, 1980. 4. Mulcahy R T, Gould M N, Hidvegi E G. Elson C E and Yatvin M B. Hyperthermia and surface morphology of P,ss ascites tumor cells: Effects of membrane modifications. Int. J. Radiat. Biol. 39: 95-106, 1981. 5. Yatvin M B. Cree T C, Elson C E, Gipp J J. Tegmo IM and Vorpahl J W. Probing the relationship of membrane “fluidity” to heat killing of cells. Radiat. Res. 89: 644-646, 1982.
AIDS THERAPY
USING HYPERTHERMIA
AND MEMBRANE MODIFICATION
165
6. Yatvin M B, Abuirmeileh N M, Vorpahl J W and Elson by low NaCl medium. J. Gen. Virol. 66: 1171-1177. C E. Biological optimization of hyperthermia: Modifi1985. cation of tumor membrane lipids. Eur. J. Cancer Oncol. 19. Lodish H’F and Kong N. Reversible block in intracell19: 657-663. lY83. ular transport and bodding of mutant vesicular stomatitis 7. Yatvin M B, Vorpahl J W. Gould M N and Lyte M. The virus glycoproteins. Virol. 125: 335-348, 1983. effects of membrane modification and hyperthermia on 20. Keough K M W and Davis P J. Thermal analysis of the survival of P,,, and V,,, cells. Eur. J Cancer Clin. membranes. pp. 55-97 in Biomembranes. Volume 12 (M Oncol. 19: 1247-1253, 1983. Kates. L A Manson, eds) Plenum Press, New York. 8 Elegbede U A, Elson C E, Qureshi A. Dennis W H and 1984. Yatv’in M 6. increasing the thermosensitivity of a .21. Cupp J. Klymkowski M. Sands J, Keith A and Snipes mammary tumor (CA,,,) through dietary modification. W. Effect of lipid alkyl chain perturbations on the Eur. J. Cancer Clin. Oncol. 22: 607-615. lY86. assembly of bacteriophage PM2. Biochim. Biophys. Acta 9 Yatvin M B. Dennis W H, Elegebe U A and Elson C E. 389: 345-357, 1975. Sensitivity of tumor cells to heat and ways of modifying 22. Snipes W. Person S. Keith A and Cupp J. Butylated the response. Sot. Exper. Biol. Symposium Series. Vol hydroxytoluene inactivates lipid-containing viruses. 41. 1987. Science 188: 64-65. 1975. 10 Yau T M. Procaine-mediated modification of membranes 23. Wanda P. Cupp J. Snipes W. Keith A. Rucinsky T, and of the response to irradiation and hyperthermia in Polish L and Sands J. Inactivation of the enveloped mammalian cells. Radiat. Res. 80: 523-541, 1979. bacteriophage 6 by butylated hydroxytoluene and butylated hydroxyanisolc. Antimicrob. Agents Chemother. II Li Ci C. Shiu E C and Hahn G M. Similarities in cellular inactivation by hyperthermia or by ethanol. Radiat. Res. IO: 96-101. 1976. 2: 757-26X, 1980. 24. Kim KS. Moon H S. Sapienza V. Carp R 1 and 12 Burns C D, Luttenegger D C. Dudley D T. Buettner Pullarkat R. Inactivation of cytomegalovirus and Semliki G R and Spector A A. Effect of modification of plasma forest virus by butylated hydroxytoluene. J. Infect. Dis. 13X: 91-03, 1978. membrane fatty acid composition on fluidity and methotrexate transport in L,,,,, murine leukemia cells. Cancer 25. Winston V D. Bolen J B and Consigli R A. Effect of butylated hydroxytoluene on Newcastle disease virus. Rcs. 39: 1726-1730, 1979. Am. J. Vet. Res. 41: 391-394. lY80. 13 Moore M F. Patzer E J. Shaw J M. Thompson T E and Wagner R R. Interaction of vesicular stomatitis virus 26. Freeman D J. Wenerstrom R N and Spruance S L. Treatment of recurrent herpes simplex labialis with with lipid vesicles: Depletion of cholesterol and effect on topical butylated hydroxtoluene. Clin. Phamacol. Ther. virion membrane fluidity and infectivity. J. Virology 27: 32Om-32’).1978. 38: 56-59. 1985. 27. Richards J T, Katz M E and Kern E R. Topical buty14 Kate\ M, Allison A C, Tyrell D A and James A T. lated hydroxytoluene treatment of genital herpes simplex Lipids of influenza virus and their relation to those of the virus infections of guinea pigs. Antiviral Research host cell. Biochim. Biophys. Acta 52: 455-466. 1961. 5: 2X1-290 (1985). 15 Pessin J E and Glaser M. Budding of Rous sarcoma virus and vesicular stomatitis virus from localized regions in 28. Pirtle E C. Sacks J M and Nachman R J. Antiviral effectiveness of butylated hydroxytoluene against pseudorathe plasma membrane of chick embryo fibroblasts. J. bies (Aujeszky’s disease) virus in cell culture. mice, and Bio. Chem. 255: 9044-9050. 1980. swine. Am. J. Vet. Res. 9: 1892-1895. 1986. 16 Heron D S. Shinitzky M and Samuel D. Alleviation of drug withdrawl symptoms by treatment with a potent 29. Reimund E. Envelope perturbation and AIDS. The mixture of natural lipids. Europ. J. Pharmacol. Lancet. Nov. IS: 1159. 1986. X3: 253-261, 1982. 30 Reimund E. Butylated hydroxytoluene. lipid-enveloped 17 Cherry R J. Modulation of protein-protein interactions viruses and AIDS. Medical Hypotheses 23: 39-42, 1987. in membranes. Biochem. Sot. Trans. 15: 91-93. 1986. 31 Lepock J R. Massicotte-Nolan P. Rule G S and Kruuv 18 Garrv R F. Bostick D A. Schram R and Waite MRF. J. Lack of a correlation between hyperthermic cell The ratio of plasma membrane cholesterol to phosphokilling. thermotolerance, and membrane fluidity. Radiat. Res. X7: 300-313, 19X1. lipid and the inhibition of Sindbis virus maturation
An Approach Hyperthermia MILTON Unwersity
to AIDS Therapy Using and Membrane Modification
B. YATVIN of Wisconsin
Medical
School,
420 N. Charter St. Madison,
WI 53706, USA.
the biophysical characteristics of cell membranes by diet and Abstract - Altering membrane perturbing agents markedly influences thermosensitivity of cells. Likewise, manipulation of viral envelopes either by altering their lipid composition by diet or by the use of agents that perturb the lipid envelope influence infectivity of enveloped viruses and the progression of viral disease. The use of hypet-thermia and envelope modification as a combined approach to treat AIDS has until now neither been suggested nor attempted. On the basis of my previous work and a review of the literature, I theorize that the combination of hyperthermia with procedures designed to alter the viral envelope will likely result in an increased viral sensitivity and be useful clinically for treatment of patients with enveloped viral diseases such as AIDS.
Introduction Over the years. my studies have led me to conclude that the initial effect of hyperthermia on cells is mediated via the heat-induced disorganization of membrane lipids. This disordering effect can be further enhanced by modifying the membrane either by diet or membrane active agents (1). I have postulated that such lipid disorganization modifies the membrane’s biophysical properties and leads to passive changes in transmembrane permeability, shifts in surface charge, and altered stereoorganization of macromolecules within the membrane (l-9). In support of the above, studies in our laboratory have shown that dietary alteration of the lipids of cell membranes has an effect on cellular thermosensitivity. and that membrane active agents
have a similar effect on the sensitivity of cells to heat (l-9). Consistent with this view are reports by others showing that treatment of mammalian cells with anaesthetics (10). alcohol (11) or highly unsaturated fatty acid diets (12) increases cell killing by hyperthermia both in vivo and in vitro. From the foregoing. it is likely that cell membranes play a critical role in determining the thermal sensitivity of cells. On the basis of the above, I propose that more effective treatment of enveloped viral disease will result when therapy includes membrane modification in conjunction with hyperthermia. Discussion
The membrane of enveloped viruses can be modified in viwo by removing cholesterol (13) or
163
164 by modifying the diet and growth conditions of the host cell both in vitro and in viuo (14, 15). When fluidity of the viral membrane was increased by removing cholesterol, infectivity of the vesicular stomatitis virus was reduced (13). Furthermore, the ordered state of membrane lipids can markedly affect protein interaction between and within membranes, including the presentation and exposure of proteins from the membrane (16) and the lateral movement of proteins within the membrane (17). Such changes in organization of cell and viral membranes may, in turn, mediate viral etiology, particularly the stages of attachment, penetration, assembly and release. For example, if viral or host cell membrane proteins are not sufficiently exposed (16), viral attachment will be inhibited. Likewise, the lateral mobility of proteins in a cell membrane (17) may affect the entry of envelope proteins into the host cell membrane and influence the assembly of the virus envelope. Viral assembly, budding and release reportedly are affected both by a low NaCI environment and by the ratio of cholesterol to phospholipid in the plasma membrane of the host cell (18). A glycoprotein involved in the maturation of a temperature-sensitive vesicular stomatitis virus did not become incorporated into the virus at a temperature of 40” C (19). At this non-permissive temperature, an appreciable amount of the viral glycoprotein reaches the plasma membrane with some being released into the medium. However, until the temperature was lowered, the viral glycoprotein remained on the cell surface and virions were not formed (19). Thus, it appears that virion assembly requires a less fluid host membrane. The above results indicate that host cell membrane organization has a major influence on the life cycle of viruses. In addition, the special lipid compound AL721 which fluidizes membranes 916), although not curative, beneficially alters the etiology of AIDS (Dr. M. Shinitzky, pers. comm.). Furthermore, there are many small hydrophobic compounds which fluidize lipid bilayers (20), some of which have already been used as antiviral agents (21-30). Of particular interest are the hydrophobic lipid perturbing molecules adamantane and butylated hydroxytoluene (BHT) and their related derivatives which have antiviral activity against enveloped viruses (21-30). The use of BHT has already been suggested as an approach to AIDS therapy in combination with reverse
MEDICAL HYPOTHESES
transcriptase inhibitors and perhaps biological response modifiers (29). The incorporation of BHT into the viral envelope causes perturbation (23) and in the case of adamantane may disrupt the envelope (21). Furthermore, when BHT is incorporated into host cell membranes, it interferes with viral budding in chicken cells (29) and adamantane affects the assembly of bacteriophage PM2 (21). In the latter case, it has been suggested that the cell membrane can function and grow with lipid alkyl chains in either the “ordered” or “disordered” state, but that the “ordered” state must be maintained for PM2 assembly to occur (21). BHT appears to have only a minimal effect on host cell membranes (30) but pretreatment of chicken embryo cells was found to inhibit the synthesis of Newcastle disease virus progeny by about 65% (25). BHT, which effectively fluidizes cell membranes of the V-,9 Chinese hamster lung fibroblasts, did not sensitize these cells to hyperthermic insult at 44.5” C (31). The above reports are consistent with idea that viral activity will be inhibited to a greater extent by membrane modification and heat that host cells. Conclusion I suggest the following as a logical approach for treating enveloped viral diseases such as AIDS: the use of hyperthermia with anaesthetics, dietary modification, or small hydrophobic compounds such as BHT and adamantane, either alone or in combination, tb maximally disrupt the viral envelope and alter the progression of viral disease. References 1. Yatvin. M B. The influence of lipid composition and procaine on hyperthermic death of cells. Int. .I. Radiat. Biol. 32: 513-521. 1977. 2. Yatvin M B, Clifton K H and Dennis W H. Hyperthermia and local anaesthetics: Potentiation of survival in tumor-bearing mice. Science 205: 195-196. 1979. 3. Hidveni E J. Yatvin M B. Dennis W H and Hidveei E. Effect” of altered membrane lipid composition -and killing of ascites cells. procaine on hyperthermic Oncology 37: 360-363, 1980. 4. Mulcahy R T, Gould M N, Hidvegi E G. Elson C E and Yatvin M B. Hyperthermia and surface morphology of P,ss ascites tumor cells: Effects of membrane modifications. Int. J. Radiat. Biol. 39: 95-106, 1981. 5. Yatvin M B. Cree T C, Elson C E, Gipp J J. Tegmo IM and Vorpahl J W. Probing the relationship of membrane “fluidity” to heat killing of cells. Radiat. Res. 89: 644-646, 1982.
AIDS THERAPY
USING HYPERTHERMIA
AND MEMBRANE MODIFICATION
165
6. Yatvin M B, Abuirmeileh N M, Vorpahl J W and Elson by low NaCl medium. J. Gen. Virol. 66: 1171-1177. C E. Biological optimization of hyperthermia: Modifi1985. cation of tumor membrane lipids. Eur. J. Cancer Oncol. 19. Lodish H’F and Kong N. Reversible block in intracell19: 657-663. lY83. ular transport and bodding of mutant vesicular stomatitis 7. Yatvin M B, Vorpahl J W. Gould M N and Lyte M. The virus glycoproteins. Virol. 125: 335-348, 1983. effects of membrane modification and hyperthermia on 20. Keough K M W and Davis P J. Thermal analysis of the survival of P,,, and V,,, cells. Eur. J Cancer Clin. membranes. pp. 55-97 in Biomembranes. Volume 12 (M Oncol. 19: 1247-1253, 1983. Kates. L A Manson, eds) Plenum Press, New York. 8 Elegbede U A, Elson C E, Qureshi A. Dennis W H and 1984. Yatv’in M 6. increasing the thermosensitivity of a .21. Cupp J. Klymkowski M. Sands J, Keith A and Snipes mammary tumor (CA,,,) through dietary modification. W. Effect of lipid alkyl chain perturbations on the Eur. J. Cancer Clin. Oncol. 22: 607-615. lY86. assembly of bacteriophage PM2. Biochim. Biophys. Acta 9 Yatvin M B. Dennis W H, Elegebe U A and Elson C E. 389: 345-357, 1975. Sensitivity of tumor cells to heat and ways of modifying 22. Snipes W. Person S. Keith A and Cupp J. Butylated the response. Sot. Exper. Biol. Symposium Series. Vol hydroxytoluene inactivates lipid-containing viruses. 41. 1987. Science 188: 64-65. 1975. 10 Yau T M. Procaine-mediated modification of membranes 23. Wanda P. Cupp J. Snipes W. Keith A. Rucinsky T, and of the response to irradiation and hyperthermia in Polish L and Sands J. Inactivation of the enveloped mammalian cells. Radiat. Res. 80: 523-541, 1979. bacteriophage 6 by butylated hydroxytoluene and butylated hydroxyanisolc. Antimicrob. Agents Chemother. II Li Ci C. Shiu E C and Hahn G M. Similarities in cellular inactivation by hyperthermia or by ethanol. Radiat. Res. IO: 96-101. 1976. 2: 757-26X, 1980. 24. Kim KS. Moon H S. Sapienza V. Carp R 1 and 12 Burns C D, Luttenegger D C. Dudley D T. Buettner Pullarkat R. Inactivation of cytomegalovirus and Semliki G R and Spector A A. Effect of modification of plasma forest virus by butylated hydroxytoluene. J. Infect. Dis. 13X: 91-03, 1978. membrane fatty acid composition on fluidity and methotrexate transport in L,,,,, murine leukemia cells. Cancer 25. Winston V D. Bolen J B and Consigli R A. Effect of butylated hydroxytoluene on Newcastle disease virus. Rcs. 39: 1726-1730, 1979. Am. J. Vet. Res. 41: 391-394. lY80. 13 Moore M F. Patzer E J. Shaw J M. Thompson T E and Wagner R R. Interaction of vesicular stomatitis virus 26. Freeman D J. Wenerstrom R N and Spruance S L. Treatment of recurrent herpes simplex labialis with with lipid vesicles: Depletion of cholesterol and effect on topical butylated hydroxtoluene. Clin. Phamacol. Ther. virion membrane fluidity and infectivity. J. Virology 27: 32Om-32’).1978. 38: 56-59. 1985. 27. Richards J T, Katz M E and Kern E R. Topical buty14 Kate\ M, Allison A C, Tyrell D A and James A T. lated hydroxytoluene treatment of genital herpes simplex Lipids of influenza virus and their relation to those of the virus infections of guinea pigs. Antiviral Research host cell. Biochim. Biophys. Acta 52: 455-466. 1961. 5: 2X1-290 (1985). 15 Pessin J E and Glaser M. Budding of Rous sarcoma virus and vesicular stomatitis virus from localized regions in 28. Pirtle E C. Sacks J M and Nachman R J. Antiviral effectiveness of butylated hydroxytoluene against pseudorathe plasma membrane of chick embryo fibroblasts. J. bies (Aujeszky’s disease) virus in cell culture. mice, and Bio. Chem. 255: 9044-9050. 1980. swine. Am. J. Vet. Res. 9: 1892-1895. 1986. 16 Heron D S. Shinitzky M and Samuel D. Alleviation of drug withdrawl symptoms by treatment with a potent 29. Reimund E. Envelope perturbation and AIDS. The mixture of natural lipids. Europ. J. Pharmacol. Lancet. Nov. IS: 1159. 1986. X3: 253-261, 1982. 30 Reimund E. Butylated hydroxytoluene. lipid-enveloped 17 Cherry R J. Modulation of protein-protein interactions viruses and AIDS. Medical Hypotheses 23: 39-42, 1987. in membranes. Biochem. Sot. Trans. 15: 91-93. 1986. 31 Lepock J R. Massicotte-Nolan P. Rule G S and Kruuv 18 Garrv R F. Bostick D A. Schram R and Waite MRF. J. Lack of a correlation between hyperthermic cell The ratio of plasma membrane cholesterol to phosphokilling. thermotolerance, and membrane fluidity. Radiat. Res. X7: 300-313, 19X1. lipid and the inhibition of Sindbis virus maturation