The effect of hypoxia and low pH on the cytotoxicity of chlorambucil

Skarsgard, L. D.; Chaplin, David J.; Wilson, Donald W.; Skwarchuk, Mark W.; Vinczan, A; Kristl, Jiri

doi:10.1016/0360-3016(92)90514-i

Cited by 12 publications

(9 citation statements)

References 9 publications

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“…Cook and Fox (1988) and Chu and Dewey (1988). Wahl et al (1996) Previous studies with non-low pH-adapted cells have shown an enhanced toxicity and/or uptake of chlorambucil at low pHe (Brophy and Sladek, 1983;Mikkelsen et al, 1985;Jahde et al, 1989;Skarsgard et al, 1992;Atema et al, 1993;Parkins et al, 1996) and the opposite effects for mitoxantrone (Jahde et al, 1990;Vukovic and Tannock, 1997). The present experimental results for both normal and acid-adapted cells are consistent with these observations.…”

Section: Resultssupporting

confidence: 88%

Cytotoxicity of weak electrolytes after the adaptation of cells to low pH: role of the transmembrane pH gradient

Kozin

Gerweck²

1998

Br J Cancer

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Summary Theory suggests that the transmembrane pH gradient may be a major determinant of the distribution of lipophilic weak electrolytes across the cell membrane. The present study evaluates the extent to which this factor contributes to pH-dependent changes in the cytotoxicity of two such chemotherapeutic drugs: chlorambucil and mitoxantrone. Experiments were performed with two cell types of the same origin but exhibiting different pH gradients at the same extracellular pH (pHe): CHO cells cultured under normal physiological conditions (pH 7.4) and acid-adapted cells obtained by culturing under low pH conditions (6.8). Over the pHe range examined (6.0-7.6), the difference between intracellular pH (pHi) and pHe increased with decreasing pHe. Acid-adapted cells were more resistant to acute changes in pHi than normal cells, resulting in substantially larger gradients in these cells. Drug cell survival curves were performed at pHe values of 6.4, 6.8 and 7.4. The cytotoxicity of chlorambucil, a weak acid, increased with decreasing pHe, and low pH-adapted cells were more sensitive than normal cells at the same pHe. In contrast, for the weak base, mitoxantrone, cytotoxicity increased with pHe and was more pronounced in normal cells. As predicted by the theory, the cytotoxicity of both drugs changed exponentially as a function of the pH gradient, regardless of cell type. For mitoxantrone, the rate of such change in cytotoxicity with the gradient was approximately two times greater than for chlorambucil. This difference is probably due to the presence of two equally ionizable crucial groups on mitoxantrone vs one group on chlorambucil. It is concluded that the cellular pH gradient plays a major role in the pH-dependent modulation of cytotoxicity in these weak electrolytes. The data obtained also suggest that a pronounced differential cytotoxicity may be expected in vivo in tumour vs normal tissue. In comparison with normal cells at a pHe of 7.4 (a model of cells in normal tissues), acid-adapted cells at a pHe of 6.8 (a model of cells distal from supplying blood vessels in tumours) were more sensitive to chlorambucil, with a dose-modifying factor of approximately 6, and were more resistant to mitoxantrone by a factor of 14.Keywords: low pH; cell adaptation; pH gradient; weak electrolytes; cytotoxicity As discussed recently (Gerweck and Seetharaman, 1996), the cellular transmembrane pH gradient is significantly decreased, or even reversed, in human solid tumours compared with the corresponding normal tissue. While the intracellular pH (pHi) is similar in both tissues (7.0-7.2 in normal and 7.1-7.3 in tumour tissue), the extracellular pH (pHe) is, on average, 0.2 pH units higher in normal tissues and 0.2-0.6 lower in tumours than pHi. This in vivo difference in cellular pH gradients provides a rationale for the selective intracellular accumulation of various lipophilic drugs, which are weak electrolytes, in tumours or normal tissues.In solution, such chemicals exhibit both a neutral and an ionized form, and the balance of...

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Section: Resultssupporting

confidence: 88%

Cytotoxicity of weak electrolytes after the adaptation of cells to low pH: role of the transmembrane pH gradient

Kozin

Gerweck²

1998

Br J Cancer

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“…The in vitro studies also confirmed the predominant role of low pHi in these potentiations by stimulated DNA alkylation of cisplatin and melphalan Atema et al, 1993) and enhanced bioreductive activation of mitomycin C and EO9 (Gibson et al, 1992;Philips et al, 1992;Begleiter and Leith, 1993). Furthermore, we confirmed that chlorambucil is exclusively potentiated at low extracellular pH conditions which, as a weak acid with a pK a value of 5.8, enhances its uptake (Mikkelsen et al, 1985;Skarsgard et al, 1992). The opposite was true for doxorubicin cytotoxicity because of a decreased uptake at low pHe conditions (Alabaster et al, 1989).…”

Section: Discussionsupporting

confidence: 75%

“…This was also in conflict with reports on enhanced cell kill and tumour response to chlorambucil simultaneously given with the vasoactive drugs hydralazine (Skarsgard et al, 1992) or flavone acetic acid (Parkins et al, 1994) to manipulate the pH. Perhaps the effect of drug trapping by a strongly reduced tumour blood flow is more important than a reduced pHe in these responses.…”

Section: Discussionmentioning

confidence: 90%

Potentiation of anti-cancer drug activity at low intratumoral pH induced by the mitochondrial inhibitor m-iodobenzylguanidine (MIBG) and its analogue benzylguanidine (BG)

et al. 1999

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Summary Tumour-selective acidification is of potential interest for enhanced therapeutic gain of pH sensitive drugs. In this study, we investigated the feasibility of a tumour-selective reduction of the extracellular and intracellular pH and their effect on the tumour response of selected anti-cancer drugs. In an in vitro L1210 leukaemic cell model, we confirmed enhanced cytotoxicity of chlorambucil at low extracellular pH conditions. In contrast, the alkylating drugs melphalan and cisplatin, and bioreductive agents mitomycin C and its derivative EO9, required low intracellular pH conditions for enhanced activation. Furthermore, a strong and pH-independent synergism was observed between the pHequilibrating drug nigericin and melphalan, of which the mechanism is unclear. In radiation-induced fibrosarcoma (RIF-1) tumour-bearing mice, the extracellular pH was reduced by the mitochondrial inhibitor m-iodobenzylguanidine (MIBG) or its analogue benzylguanidine (BG) plus glucose. To simultaneously reduce the intracellular pH, MIBG plus glucose were combined with the ionophore nigericin or the Na + /H + exchanger inhibitor amiloride and the Na + -dependent HCO 3 -/Cl -exchanger inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulphonic acid (DIDS). Biochemical studies confirmed an effective reduction of the extracellular pH to approximately 6.2, and anti-tumour responses to the interventions indicated a simultaneous reduction of the intracellular pH below 6.6 for at least 3 h. Combined reduction of extra-and intracellular tumour pH with melphalan increased the tumour regrowth time to 200% of the pretreatment volume from 5.7 ± 0.6 days for melphalan alone to 8.1 ± 0.7 days with pH manipulation (P < 0.05). Mitomycin C related tumour growth delay was enhanced by the combined interventions from 3.8 ± 0.5 to 5.2 ± 0.5 days (P < 0.05), but only in tumours of relatively large sizes. The interventions were non-toxic alone or in combination with the anti-cancer drugs and did not affect melphalan biodistribution. In conclusion, we have developed non-toxic interventions for sustained and selective reduction of extra-and intracellular tumour pH which potentiated the tumour responses to selected anti-cancer drugs.

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“…Additionally, the choice of the drugs to evaluate the effect of pH gradient manipulation should exhibit the following characteristics: pK a values in the physiologic pH range, an intracellular site of action and entry to the site of action by passive diffusion through the cell membrane, equivalent cytotoxicity under oxygenated and hypoxic conditions, and stability under relevant pH conditions. The weak acid chlorambucil and weak base doxorubicin meet these criteria (6,12,22,23).…”

Section: Introductionmentioning

confidence: 99%