Tuftsin augments antitumor efficacy of liposomized etoposide against fibrosarcoma in Swiss albino mice

Abstract

Anticancer drugs are generally plagued by toxic manifestations at doses necessary for control of various forms of cancer. Incorporating such drugs into liposomes not only reduces toxicity but also enhances the therapeutic index. Some antioxidants and potent immunomodulators have also been shown to impart significant antitumor activity presumably by nonspecific activation of the host immune system. In the present study, we evaluated augmentation of the antitumor activity of etoposide (ETP) by the immunomodulator tuftsin in Swiss albino mice with fibrosarcoma. The efficacies of the free form of ETP, liposomized ETP (Lip-ETP), and tuftsin-bearing liposomized ETP (Tuft-Lip-ETP) formulations were evaluated on the basis of tumor regression, effect on expression level of p53wt and p53mut, and survival of the treated animals. Tuft-Lip-ETP, when administered at a dosage of 10 mg/kg body weight/day for five days, significantly reduced tumor volume, delayed tumor growth, and also up-regulated the expression of p53wt. In contrast, although Lip-ETP delayed tumor growth, it did not decrease tumor size. The results of the present study suggest that tuftsin incorporation in drug-loaded liposomes is a promising treatment strategy for various forms of cancers, including fibrosarcoma.

Figure 1

(A) Leakage of ETP from liposomized formulations in surrounding PBS buffer. Liposomized formulations of ETP were incubated in PBS for 24 h and 48 h. Aliquots were taken from the supernatant to determine released ETP by HPLC as described in Materials and Methods. Data are mean ± SD of 3 independent experimental values. *P < 0.001 (Tufts-Lip-ETP) versus Lip-ETP at 24 h and 48 h. (B) Hemolysis of human erythrocytes induced by various formulations of ETP. Human erythrocytes were incubated with free ETP, Lip-ETP, and Tuft-Lip-ETP for 1 h at 37 °C as described in Materials and Methods. Data are mean ± SD of 3 independent experimental values. Various groups were compared by one-way ANOVA followed by Dunnett’s post hoc test. ^aP < 0.001 versus free ETP; ^bP < 0.001 versus Lip-ETP.

Figure 2

Effect of increasing doses of ETP on body weight of the treated animals. The Swiss mice were treated with free ETP, Lip-ETP, and Tuft-Lip-ETP. The dose resulting in the loss of more than 10% body weight is identified as the MTD. Lip-ETP had a significantly higher MTD than free ETP, and Tuft-Lip-ETP had a significantly higher MTD than Lip-ETP. Data shown are means of three individual animals at each dose point.

Figure 3

Effects of ETP chemotherapy on leukocyte and platelet counts of the treated animals. Experimental animals were treated with various formulations of ETP. The blood samples of the treated animals were analyzed for their lymphocyte and platelet counts. (A) Free ETP significantly reduced the total leukocyte count (P < 0.001) compared with controls. Lip-ETP caused lesser myelosuppression compared with free ETP (P < 0.001). Tuft-Lip-ETP depleted fewer leucocytes compared with free ETP (P < 0.001) and Lip-ETP (P < 0.001). (B) A similar effect was observed on platelet counts as seen for total leukocyte count after treatment with free ETP (P < 0.001). The Lip-ETP group had significantly higher mean platelet counts (P < 0.001) than the free ETP group. The Tuft-Lip-ETP group had even higher platelet counts than the free (P < 0.001) and Lip-ETP (P < 0.001) groups. Data are means ± SD with n = 5 per group. ^aP < 0.001 versus free ETP; ^bP < 0.001 versus Lip-ETP.

Figure 4

Establishment of fibrosarcoma by exposure of animals to benzo (a) pyrene. Administration of benzo (a) pyrene (250 μg/animal, subcutaneously) into the flanks of the animals resulted in successful tumor induction in at least 90 % of the animals and no tumor development in 10 % of the animals. The tumor-bearing animals were killed by cervical dislocation and fixed in 10 % formaldehyde solution. Subsequently tissues were excised and sections were prepared after standard HE staining of 12-mm-thin sections. (A) Photomicrograph from tumor showing zone of necrosis, increased vascularization and hemorrhage. Large numbers of hyperchromatic mesenchymal cells with features of fibrosarcoma are present. Some hyperchromatic cells are arranged in glandular form. (B) Photomicrograph from tumor showing soft tissue sarcoma with large pleomorphic, hyperchromatic spindle-shaped cells with anisonucleosis and irregularly dispersed chromatin. Some inflammatory cells can also be seen.

Figure 5

(A) Effects of chemotherapy with various formulations of ETP on tumor development in Swiss mice. Treatment of tumor-bearing animals was started when the tumor size reached a volume of approximately 200 mm³. Free ETP and sham tuftsin liposomes significantly delayed tumor growth (P < 0.05) as compared with controls (PBS and Sham liposomes). Liposomal ETP was superior to free ETP in delaying tumor growth (P < 0.001). The regressions of tumors were recorded only in the group of animals treated with Tuft-Lip-ETP. Data are values ± SD (n = 20 at initiation of therapy, the number varies at later time points due to mortality). (B) Effects of ETP chemotherapy on the survival of tumor-bearing mice. The fibrosarcoma was induced by exposure to benzo (a) pyrene. The treatment of tumor-bearing animals was started at the time point when tumor size reached a volume of approximately 200 mm³. All the treated mice received a dose of 10 mg/kg body weight/day for 5 d with various formulations of the drug. Free ETP significantly enhanced the life span of the mice compared with the control group (PBS and sham liposomes) (P < 0.01). Lip-ETP was superior to free ETP in increasing the lifespan (P < 0.001). The best therapeutic effect was recorded in the group of animals treated with Tuft-Lip-ETP (P < 0.001). Data shown are the number of mice surviving at the given time points.

Figure 6

(A) Effect of various formulations on the expression of p53wt in mouse fibrosarcoma. Skin/tumor lysates were prepared as described in Materials and Methods and 30–50 μg protein was used for Western blot analysis. A diminutive expression of p53wt was recorded in the animals treated with vehicle controls. Treatment with various formulations was found to replenish the p53wt expression in fibrosarcoma. Lane 1, untreated (Normal); lane 2, vehicle control (PBS); lane 3, sham egg PC liposomes; lane 4, Sham-Tuft-Lip; lane 5, free ETP; lane 6, Lip-ETP; and lane 7, Tuft-Lip-ETP. (B) Effect of various formulations on the expression of mutp53 in mouse fibrosarcoma. Skin/tumor lysates were prepared as described in Materials and Methods and proteins (30–50 μg) were employed for Western blots. The expression of p53mut was induced significantly in the animals treated with vehicle controls; however, treatment with various formulations against fibrosarcoma resulted in a decrease in p53mut expression. Lane 1, untreated (normal); lane 2, vehicle control (PBS); lane 3, sham liposomes; lane 4, Sham-Tuft-Lip; lane 5, free ETP; lane 6, Lip-ETP; and lane 7, Tuft-Lip-ETP.