Experimental Chemotherapy © 1990 S. Karger AG. Basel 0009-3157/90/0363-0230 $ 2.75/0
Chemotherapy 1990;36:230-239
Combined Effect in vitro of Chemotherapy with Agents Acting on the Cell Membrane of Lewis Lung Carcinoma Annette Siegal, Shoshana Hoenig, Judith Leibovici Departmenf of Pathology, Sackler Faculty of Medicine, Tel Aviv University, Israel
Key Words. Drug permeability • Drug resistance • Adriamycin • Hyperthermia • Verapamil ■Lewis lung carcinoma
Introduction
Despite considerable advance reached during the last decades in cancer treat ment, the cure of epithelial malignancies, most particularly in their late stages, has not yet been achieved. The mass of tumor at these stages of the disease as well as the difficulty of drugs to reach widespread growth are important factors in the failure of therapy of ad
vanced cancer. However, a relatively newly described phenomenon, the emer gence of drug-resistant clones in the tumor cell population [1-4] appears to constitute a major reason for the failure of metastatic disease treatment. Therefore, one of the aims of the search of new treatment mo dalities against cancer in its late stages is to try to circumvent tumor cell resistance to cytotoxic agents. One of the most important mechanisms
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/31/2018 6:03:11 AM
Abstract. The treatment of cancer is often impeded by the emergence of drug-resis tant clones. Drug resistance is in many cases due to a decreased drug accumulation in the tumor cell. Membrane-acting agents, by increasing cell permeability, might counter act the loss of sensitivity to drugs. In the present study, the effect of two membrane-act ing agents, hyperthermia and the calcium channel blocker verapamil, on the action of adriamycin (ADR) on Lewis lung carcinoma (3LL) was examined. Hyperthermia in creased drastically the antitumoral effect of ADR. The effectiveness of the combined ADR-hyperthermia treatment was proportional to the ADR dose and to the degree of hyperthermia. Verapamil had a similar effect but at higher ADR doses. Treatment mo dalities designed for the circumvention of drug resistance could be one approach in the attempt to find a way to control cancer.
231
Cytotoxic and Membrane-Acting Agent Effect on 3LL Cells
Materials and Methods Animals and Tumor C57BL male mice, 6-10 weeks old, were obtained from the Animal Breeding Center of the Tel Aviv University. The Lewis lung carcinoma was main tained by subcutaneous transfer. In experiments, 2 x 105 viable cells, prepared as previously described [37], were inoculated subcutaneously in the back of mice in 0.2 ml RPMI medium. Each experimental group consisted of 5 mice. Tumor growth was evalu ated by recording the incidence and size (average of two perpendicular diameters in mm) of tumors about 3 times/week. Mortality of mice was recorded daily. In vitro Thermochemotherapeutic Treatment of Lewis Lung Carcinoma Cells Hyperthermic in vitro treatment of tumor cells, alone and in combination with adriamycin (ADR) purchased from Farmitalia, Italy, was done as fol lows: Lewis lung carcinoma cells (l06/ml in RPMI medium) were incubated in thermostated waterbaths (± 0.2 °C) for 1 h at 37 °C and at hyperthermic temper atures of 42 and 43°C, in the presence or absence of ADR (5, 7.5 and 10 pg/ml). After incubation, the treated tumor cells were tested for tumorigenicity fol lowing subcutaneous inoculation to mice as de scribed above. Verapamil Treatment Verapamil was used at a concentration of 20 p Af, alone or in combination with ADR (5 and 10 pg/ml). Treatment was done at 37 °C during 1 h. After incu bation tumorigenic capacity was tested as above. Statistical Evaluation Statistical evaluation was done by Student’s t test.
Results Figures 1 and 2 present the effect of ADR at two concentrations (5 and 7.5 pg/ ml, respectively), alone and in combina tion with hyperthermia (42 °C), on the tu morigenic capacity of Lewis lung carci noma cells as evaluated by tumor size. While treatment at 42 °C alone did not re-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/31/2018 6:03:11 AM
of drug resistance evolution is a decreased cell permeability to the drug [5, 6]. Cell membrane alterations have been found to be responsible for the change in cell per meability [7-9]. Augmentation of cyto toxic drug uptake by membrane-acting agents could therefore be considered as a possibility of counteracting drug resist ance. In the present study, the effects of two membrane-acting agents, hyperthermia and verapamil, on the adriamycin effect on Lewis lung carcinoma cells was ex amined. One of the mechanisms of the antineoplastic effect of hyperthermia is an action on the cell membrane [10-18]. Hy perthermic treatment has been found to enhance the inhibitory effects of various cytotoxic agents on different tumors [19-23]. Verapamil acts on the cell mem brane as a calcium channel blocker [24-26], Increased activity of antitumoral cytotoxic agents in the presence of calcium antagonists has been reported [27-35]. Al though the molecular basis of the action of Ca2+ blockers has not yet been elucidated, it has been suggested that since they are lipophilic, they probably produce confor mational changes in the cell membrane [25], Verapamil [33] and another Ca++ channel blocker, flunarizine [35] have been found to increase tumor cell permea bility to a cytotoxic agent. Hyperthermia also affects the lipid constituent of the cell membrane [11, 15, 16, 18]. It has been sug gested that it may as well affect the cal cium channels [36]. The use of membrane-acting agents in the presence of chemotherapeutic drugs may possibly enhance their antineoplastic capacity, eventually enabling them to act on resistant tumor cell subpopulations.
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Fig. 2. Effect of ADR (7.5 pg/m l) and hyperthermia (42 °C) singly and in combined treatment on tumori genicity of Lewis lung carcinoma cells: size of tumors.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/31/2018 6:03:11 AM
Fig. 1. Effect of ADR (5 pg/ml) and hyperthermia (42 °C) singly and in combined treatment on Lewis lung carcinoma cell tumorigenicity: size of tumors.
Cytotoxic and Membrane-Acting Agent Effect on 3LL Cells
233
Fig. 4. Effect of ADR (10 pg/ml) and hyperthermia (43 °C) singly and in combined treatment on tumorigenicity of Lewis lung carcinoma cells: size of tumors.
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Fig. 3. Effect of ADR (5 pg/ml) and hyperthermia (43 °C) singly and in combined treatment on tumorigenicity of Lewis lung carcinoma cells: size of tumors.
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Siegal/Hoenig/Leibovici
Fig. 5. Effect of ADR (5 pg/ml) and hyperthermia (42
Chemotherapy 1990;36:230-239
Combined Effect in vitro of Chemotherapy with Agents Acting on the Cell Membrane of Lewis Lung Carcinoma Annette Siegal, Shoshana Hoenig, Judith Leibovici Departmenf of Pathology, Sackler Faculty of Medicine, Tel Aviv University, Israel
Key Words. Drug permeability • Drug resistance • Adriamycin • Hyperthermia • Verapamil ■Lewis lung carcinoma
Introduction
Despite considerable advance reached during the last decades in cancer treat ment, the cure of epithelial malignancies, most particularly in their late stages, has not yet been achieved. The mass of tumor at these stages of the disease as well as the difficulty of drugs to reach widespread growth are important factors in the failure of therapy of ad
vanced cancer. However, a relatively newly described phenomenon, the emer gence of drug-resistant clones in the tumor cell population [1-4] appears to constitute a major reason for the failure of metastatic disease treatment. Therefore, one of the aims of the search of new treatment mo dalities against cancer in its late stages is to try to circumvent tumor cell resistance to cytotoxic agents. One of the most important mechanisms
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/31/2018 6:03:11 AM
Abstract. The treatment of cancer is often impeded by the emergence of drug-resis tant clones. Drug resistance is in many cases due to a decreased drug accumulation in the tumor cell. Membrane-acting agents, by increasing cell permeability, might counter act the loss of sensitivity to drugs. In the present study, the effect of two membrane-act ing agents, hyperthermia and the calcium channel blocker verapamil, on the action of adriamycin (ADR) on Lewis lung carcinoma (3LL) was examined. Hyperthermia in creased drastically the antitumoral effect of ADR. The effectiveness of the combined ADR-hyperthermia treatment was proportional to the ADR dose and to the degree of hyperthermia. Verapamil had a similar effect but at higher ADR doses. Treatment mo dalities designed for the circumvention of drug resistance could be one approach in the attempt to find a way to control cancer.
231
Cytotoxic and Membrane-Acting Agent Effect on 3LL Cells
Materials and Methods Animals and Tumor C57BL male mice, 6-10 weeks old, were obtained from the Animal Breeding Center of the Tel Aviv University. The Lewis lung carcinoma was main tained by subcutaneous transfer. In experiments, 2 x 105 viable cells, prepared as previously described [37], were inoculated subcutaneously in the back of mice in 0.2 ml RPMI medium. Each experimental group consisted of 5 mice. Tumor growth was evalu ated by recording the incidence and size (average of two perpendicular diameters in mm) of tumors about 3 times/week. Mortality of mice was recorded daily. In vitro Thermochemotherapeutic Treatment of Lewis Lung Carcinoma Cells Hyperthermic in vitro treatment of tumor cells, alone and in combination with adriamycin (ADR) purchased from Farmitalia, Italy, was done as fol lows: Lewis lung carcinoma cells (l06/ml in RPMI medium) were incubated in thermostated waterbaths (± 0.2 °C) for 1 h at 37 °C and at hyperthermic temper atures of 42 and 43°C, in the presence or absence of ADR (5, 7.5 and 10 pg/ml). After incubation, the treated tumor cells were tested for tumorigenicity fol lowing subcutaneous inoculation to mice as de scribed above. Verapamil Treatment Verapamil was used at a concentration of 20 p Af, alone or in combination with ADR (5 and 10 pg/ml). Treatment was done at 37 °C during 1 h. After incu bation tumorigenic capacity was tested as above. Statistical Evaluation Statistical evaluation was done by Student’s t test.
Results Figures 1 and 2 present the effect of ADR at two concentrations (5 and 7.5 pg/ ml, respectively), alone and in combina tion with hyperthermia (42 °C), on the tu morigenic capacity of Lewis lung carci noma cells as evaluated by tumor size. While treatment at 42 °C alone did not re-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/31/2018 6:03:11 AM
of drug resistance evolution is a decreased cell permeability to the drug [5, 6]. Cell membrane alterations have been found to be responsible for the change in cell per meability [7-9]. Augmentation of cyto toxic drug uptake by membrane-acting agents could therefore be considered as a possibility of counteracting drug resist ance. In the present study, the effects of two membrane-acting agents, hyperthermia and verapamil, on the adriamycin effect on Lewis lung carcinoma cells was ex amined. One of the mechanisms of the antineoplastic effect of hyperthermia is an action on the cell membrane [10-18]. Hy perthermic treatment has been found to enhance the inhibitory effects of various cytotoxic agents on different tumors [19-23]. Verapamil acts on the cell mem brane as a calcium channel blocker [24-26], Increased activity of antitumoral cytotoxic agents in the presence of calcium antagonists has been reported [27-35]. Al though the molecular basis of the action of Ca2+ blockers has not yet been elucidated, it has been suggested that since they are lipophilic, they probably produce confor mational changes in the cell membrane [25], Verapamil [33] and another Ca++ channel blocker, flunarizine [35] have been found to increase tumor cell permea bility to a cytotoxic agent. Hyperthermia also affects the lipid constituent of the cell membrane [11, 15, 16, 18]. It has been sug gested that it may as well affect the cal cium channels [36]. The use of membrane-acting agents in the presence of chemotherapeutic drugs may possibly enhance their antineoplastic capacity, eventually enabling them to act on resistant tumor cell subpopulations.
232
Siegal/Hoenig/Leibovici
Fig. 2. Effect of ADR (7.5 pg/m l) and hyperthermia (42 °C) singly and in combined treatment on tumori genicity of Lewis lung carcinoma cells: size of tumors.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/31/2018 6:03:11 AM
Fig. 1. Effect of ADR (5 pg/ml) and hyperthermia (42 °C) singly and in combined treatment on Lewis lung carcinoma cell tumorigenicity: size of tumors.
Cytotoxic and Membrane-Acting Agent Effect on 3LL Cells
233
Fig. 4. Effect of ADR (10 pg/ml) and hyperthermia (43 °C) singly and in combined treatment on tumorigenicity of Lewis lung carcinoma cells: size of tumors.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/31/2018 6:03:11 AM
Fig. 3. Effect of ADR (5 pg/ml) and hyperthermia (43 °C) singly and in combined treatment on tumorigenicity of Lewis lung carcinoma cells: size of tumors.
234
Siegal/Hoenig/Leibovici
Fig. 5. Effect of ADR (5 pg/ml) and hyperthermia (42
