Efficacy of Imiquimod for the Expression of Bcl-2, Ki67, p53 and Basal Cell Carcinoma Apoptosis

D. Vidal; X. Matías-Guiu; A. Alomar

Disclosures

The British Journal of Dermatology. 2004;151(3) 

In This Article

Summary and Introduction

Background: Imiquimod is a modifier of the immune response that has been proven to be an effective treatment for basal cell carcinoma (BCC). However, its mechanism of action is still unknown.
Objectives: To determine whether imiquimod modifies the expression of proteins such as Bcl-2, Ki67, p53 and the BCC apoptotic index.
Patients and Methods: Thirty caucasian patients with primary BCCs larger than 8 mm in diameter were included in a double-blind randomized clinical and immunohistochemical study which was designed in a reference university hospital. The 30 BCCs were randomized in two treatment arms between September 2001 and February 2002. Twenty-four BCCs were treated with imiquimod 5% cream and six BCCs with Aldara® (3M Pharmaceuticals) excipient. Histological samples were obtained before treatment and on days 8 and 15 during the course of treatment. The BCC expression of Bcl-2, Ki67 and p53 was determined in paraffin samples and the apoptotic index of the BCC was studied using the TUNEL technique (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end labelling) in frozen samples. All variables were evaluated quantitatively in fields with a magnification × 400.
Results: The BCCs treated with imiquimod showed a decrease in the expression of Bcl-2 (88·7% before treatment, 61·4% day 15, P = 0·01) and an increase in the apoptotic index (0·53% before treatment, 1·66% day 15, P = 0·002), which were not observed in the BCCs treated with the excipient. Ki67 and p53 did not show significant changes in any group.
Conclusions: Imiquimod reduces the expression of Bcl-2 in the BCC cells and increases the BCC apoptotic index.

Basal cell carcinoma (BCC) is the most frequent malignant tumour in the white human population worldwide. Its incidence is increasing worldwide due to ageing of the population and chronic sun exposure. BCC treatment is based on surgery. However, topical immunotherapy with imiquimod 5% cream is an emergent therapeutic option that enables the outpatient treatment of BCC. Imiquimod is a synthetic molecule of 240·3 Da [C14 H16 N4, 1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine]. In vitro and in vivo studies have proven that imiquimod is a cytokine inductor and a modifier of the innate immune response, as well as acquired antiviral and antitumour immune response. Imiquimod efficacy proved to be effective in superficial and nodular BCCs in five randomized trials with placebo.[1,2,3,4,5] The mechanism of action of imiquimod in the treatment of BCC is still unknown. However, some clinical studies[6] have proven that imiquimod leads to an acute immune response modifying the peritumoral inflammatory infiltrate and increasing CD4+ T lymphocytes. The main cytokine induced by imiquimod is interferon-alfa (IFN-α). Therefore, some hypotheses on the mechanism of action of imiquimod in BCC are based on this cytokine. Intralesional IFN-α proved to be effective in BCC[7] and its mechanism of action is based on causing an antitumour inflammatory infiltrate, intensifying the expression of the tumour antigens, inhibiting cell proliferation and increasing apoptosis of BCC cells.[8,9]

Apoptosis, which is programmed cell death or cellular suicide, is a physiological process by which nonviable cells are eliminated. Apoptosis acts as a basic cellular process in the regulation of the cell population, both in the morphogenesis and homeostasis of tissues. Apoptosis is a strictly regulated process and many genes and proteins participate in its control. At the same time, most of the genes involved in the control of apoptosis participate in the mechanisms regulating the cellular cycle. The alteration of these genes may lead to carcinogenesis.[10,11] The TUNEL technique[12] (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end labelling) is a complex immunohistochemical technique,[13] but positive cells in the TUNEL technique are considered to be apoptotic cells. The apoptotic index for BCC is higher than for normal skin, and this fact may explain the slow progression of BCC.[14] Aggressive BCC presents higher proliferation and apoptotic indices than nonaggressive BCC.[15]

Bcl-2 is a proto-oncogene that encodes the largest known antiapoptotic protein. Bcl-2 regulates apoptosis through the formation of homodimer and heterodimer proteins with the other proteins belonging to the Bcl-2 group, especially the proapoptotic protein Bax.[16] A nonaggressive BCC expresses more Bcl-2 than an aggressive BCC.[17] BCC expresses very little Bax,[18] so that the Bcl-2 apoptotic effect is predominant and tumour progression is promoted. Ki67 is a nonhistone protein that is detected in the nucleus in the phases G1, S, G2 and M of the cell cycle, but not in phase G0.[19] The number of cells with Ki67 is used to mark cell proliferation. Ki67 expression is higher in aggressive and recurring BCC.[20] p53 is a tumour suppressor gene, which is known as the guardian of the genome. This is because of its ability to regulate the cell cycle in stressful situations and to keep genome integrity.[21] When there is any alteration to the p53 genome, p53 activates the p21 pathway. The latter inhibits CDK2 and stops the cell cycle in phase G1, enabling the actuation of the DNA repair mechanisms before phase S. On the other hand, when the DNA mutations are irreversible, p53 activates the Bax pathway, which leads to the apoptosis.

The objectives of this study were to determine whether imiquimod reduces the antiapoptotic protein Bcl-2 or tumour cell proliferation (Ki67 expression), and also whether it modifies protein p53 expression and increases the BCC apoptotic index.

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