Potential of diallyl sulfide bearing pH-sensitive liposomes in chemoprevention against DMBA-induced skin papilloma

Abstract

Diallyl sulfide (DAS), an active component of garlic, possesses strong anti-neoplastic properties against various forms of cancer. In the present study, we have evaluated chemo-preventive effects of liposomized DAS (conventional egg PC and pH-sensitive liposomes) against DMBA-induced skin papilloma. Various liposome-based novel formulations of DAS (250 microg/mouse) were applied topically, after one hour of exposure to DMBA (52 microg/mouse/dose), to the animals. The animals were treated thrice weekly for the total period of 12 weeks. The efficacy of the various liposomal formulations of DAS was evaluated on the basis of parameters such as incidence of tumorogenesis and total numbers and sizes of induced tumor nodules. The liposomized DAS formulations also were assessed for their effect on the expression of p53wt, p53mut, and p21/Waf1. The results of the present study showed that liposomized DAS could effectively delay the onset of tumorogenesis and reduce the cumulative numbers and sizes of tumor papillomas in treated mice. Treatment of DMBA-exposed animals with the liposomal formulation of DAS ensued in upregulation of p53wt and p21/Waf1, while levels of p53mut expression reduced down. The promising chemo-preventive nature of liposomal DAS may form the basis for establishing effective means of controlling various forms of cancer, including skin papilloma.

Figure 1

Chemical structure of DAS

Figure 2

Chemo-preventive effect of various formulations of DAS on onset of mouse skin tumorogenesis. The animals were exposed to DMBA and subsequently treated with various forms of DAS as described in Materials and Methods. The onset of tumorogenesis was recorded as the first appearance of a DMBA-induced tumor in each group of animals. ^aP < 0.01 (free DAS), ^bP < 0.001 (Lip-DAS and pH-Lip-DAS) versus vehicle control groups (groups II–V), ^cP < 0.001 (pH-Lip-DAS and Lip-DAS) versus free DAS (group VI).

Figure 3

A. Effect of liposomized DAS mediated chemo-prevention on the development of average number of tumors per mouse. The animals were treated with various forms of DAS after their exposure to carcinogenic agent DMBA. Efficacy of various DAS liposomal formulations was assessed on the basis of their ability to prevent development of tumors. ^aP < 0.001 (free DAS, Lip-DAS, pH-Lip-DAS) versus vehicle control groups (groups II–V), ^bP < 0.001 (Lip-DAS and pH-Lip-DAS) versus free DAS, ^cP < 0.01 (pH-Lip-DAS) versus Lip-DAS. B. Chemo-preventive effects of various formulations of DAS on average tumor size. The chemo-preventive efficacy of various forms of DAS was assessed by measuring the size of the tumors using a caliper. The tumor volume was determined by the formula as described in Materials and Methods. ^aP < 0.001 versus vehicle controls (groups II–V), ^bP < 0.001 (Lip-DAS and pH-Lip-DAS) versus free DAS. ^cP < 0.001 (pH-Lip-DAS) versus Lip-DAS (group VII). C. Percent-inhibition of tumor growth by various formulations of DAS. The animals were treated with various forms of DAS after exposure to DMBA as described in Materials and Methods. The tumor growth inhibition was calculated by comparing the average size of the tumor induced in animals that were treated with vehicle control (acetone treatment). ^aP < 0.001 versus (Free DAS). ^bP < 0.001 versus Lip-DAS.

Figure. 4

Effect of various formulations of DAS on survival of tumor-free animals. Kaplan-Meier curve showing effect of various formulations of DAS in terms of percentage of animals free from tumor burden at different time intervals. The study was scheduled for a period of 12 weeks as described in Materials and Methods. The analysis was made on weekly basis.

Figure 5

A. Effect of various formulations of DAS on the expression of p53wt in mouse skin tumors. Skin/tumor lysates were prepared as described in Materials and Methods. Lysates were resolved by electrophoresis and analyzed for p53wt using anti-p53wt antibodies. To quantify equal loading, membranes were re-probed with β-actin antibody. The intensity of the bands was quantitated using Image Analysis software on an Image Gel Documentation System. Lane 1, untreated; lane 2, DMBA + Acetone; lane 3, DMBA + Cream; lane 4, DMBA + Sham-Lip; lane 5, DMBA + Sham-pH-Lip; lane 6, DMBA + Free DAS; lane 7, DMBA + Lip-DAS; lane 8, DMBA + pH-Lip-DAS and lane 9, Cream base only with no DMBA. B. Effect of various formulations of DAS on the expression of p53mut in mouse skin tumors. Skin/tumor lysates were prepared as described in Materials and Methods and analyzed for p53mut expression by Western blot analysis using antibody to p53mut. To quantify equal loading, membranes were reprobed with β-actin antibody. The intensity of the bands was quantitated using Image Analysis software on an Image Gel Documentation System. Lane 1, untreated; lane 2, DMBA + Acetone; lane 3, DMBA + Cream; lane 4, DMBA + Sham-Lip; lane 5, DMBA + Sham-pH-Lip; lane 6, DMBA + Free DAS; lane 7, DMBA + Lip-DAS; lane 8, DMBA + pH-Lip-DAS and lane 9, Cream base only with no DMBA.

Figure 6

Effect of various formulations of DAS on the expression of p21/Waf1 in mouse skin tumors. Skin/tumor lysates were prepared as described in Materials and Methods and proteins samples from various groups were employed for Western blots. The level of p21/waf1 was assessed by Western blotting using anti-p21/Waf1 antibodies. To quantify equal loading, membranes were reprobed with β-actin antibody. The intensity of the bands was quantitated using Image Analysis software on an Image Gel Documentation System. Lane 1, untreated; lane 2, DMBA + Acetone; lane 3, DMBA + Cream; lane 4, DMBA + Sham-Lip; lane 5, DMBA + Sham-pH-Lip; lane 6, DMBA + Free DAS; lane 7, DMBA + Lip-DAS; lane 8, DMBA + pH-Lip-DAS and lane 9, Cream base only with no DMBA.