Elsevier

Biochemical Pharmacology

Volume 69, Issue 9, 1 May 2005, Pages 1343-1350
Biochemical Pharmacology

The effect of quercetin on pro-apoptotic activity of cisplatin in HeLa cells

https://doi.org/10.1016/j.bcp.2005.01.022Get rights and content

Abstract

It is well known that some tumour cells are very resistant to chemotherapy-induced cell death which indicate poor prognosis for patients. Thus the aim of the present study was to investigate the effect of quercetin on pro-apoptotic activity of cisplatin in human cervix carcinoma cells (HeLa).

Three variants of experiments were performed. In the first one cells were incubated with studied drugs separately for 8 and 24 h. In the second, drugs were added to the culture medium simultaneously. In third cisplatin or quercetin addition was followed by subsequent quercetin or cisplatin treatment, respectively.

We observed different apoptotic effects, dependent on the drug succession. Preincubation of cells with quercetin followed by cisplatin treatment appeared to be the most effective and was correlated with strong activation of caspase-3 and inhibition of both heat shock proteins (Hsp72) and multi-drug resistance proteins (MRP) levels.

Our results indicate that quercetin pretreatment sensitizes HeLa cells to cisplatin-induced apoptosis in HeLa cells.

Introduction

Apoptosis is a physiological process, which occurs during embryonic development as well as during maintenance of tissue homeostasis, tumour development and metastasis. It can be induced by a variety of treatments like UV irradiation and chemotherapeutic drugs [1], [2]. The transduction and execution of apoptotic signals requires coordinated activation of the cascade of caspases (cysteine proteases) [3], [4]. One of the critical enzyme is caspase-3. It belongs to the CED-3 subfamily and specifically cleave substrates including the poly-(ADP-ribose) polymerase (PARP) or inhibitor of caspase-activated DNase (ICAD). It also plays a major role in the chromatin condensation or DNA fragmentation [5].

It is well known that some tumour cells are very resistant to chemotherapy-induced cell death which indicate poor prognosis for patients [6]. It has been reported that cisplatin (cis-dichlorodiammine-platinum (II) CDDP) may induce apoptosis through caspase-3-dependent and -independent signal pathways [5], [7]. Cisplatin is widely used chemotherapeutic agent in the treatment of several malignances like ovarian, testicular, bladder, lung, head and neck cancers. It interacts with cellular proteins and lipids and forms DNA adducts causing cell cycle arrest in G2/M phase [8], [9], [10]. It can also disturb cytoskeleton organization [11]. However, drug resistance compromise its clinical effectiveness. It has been shown that alteration in caspase-3 expression and activity is associated with the development of cellular resistance to cisplatin. It has also been shown that the mechanism of the relative resistance to apoptosis may be connected with reduced cytochrome c release and PARP cleavage [5], [7]. There are also several other factors responsible for such resistance. One of them are heat shock proteins. These are molecular chaperones, controlling the proper folding of newly synthesized polypeptides, the refolding of missfolded proteins, and controlling translocation through cell membranes [12], [13], [14], [15]. Their expression can be modulated by factors leading to apoptosis in such pathologic states as ischemia, fever, inflammation, infections and cancers [16], [17]. The enhanced expression of Hsps has been reported for nearly all classes of tumours [6], [18], [19], [20]. Therefore, tumour cells resistance to apoptosis is thought to be closely associated with Hsps overexpression. This may also indicate poor prognosis. Thus decreasing Hsps level in cancer cells would be beneficial for patients.

Tumour cells resistance to cisplatin treatment may be also connected with multi-drug resistance proteins expression. MRPs are an integral membrane P-glycoprotein which belongs to the family of the ATP-binding casette (ABC) of transmembrane transporter proteins. They expel cytotoxic drugs and diminish their concentration to intercellular level which is not lethal for cells in consequence [7].

It was previously shown that antiproliferative effect of cisplatin was enhanced by quercetin in vivo and in vitro [21], [22]. Quercetin (3,3′,4′,5,7-pentahydroksyflavone) belongs to flavonoids. It is present in many edible fruits and vegetables like: apples, grapes, lemons, onion, kale, tomatoes. The daily intake of it is estimated to be about 25 mg. Quercetin has a broad range of biological, pharmacological and medical applications [23], [24], [25]. It has been reported to be an anticancer agent because it exerts antiproliferative effects on different malignant cells by several mechanisms: freezing the cell cycle in control points G0/G1, G1/S and G2/M, interaction with estrogen II binding sites, inhibiting glycolysis and activity of several enzymes like protein kinase C (PKC), mitogen-activated protein (MAP) kinase, cell division cycle (CDC) kinase 2, tyrosine kinases, phosphoinositol-3, -4 and -5 kinases. It diminishes the expression of genes necessary for cell proliferation like: H-ras, K-ras, N-ras i c-myc [26], [27], [28], [29], [30], [31], [32], [33], [34], [35]. Like other members of flavonoids quercetin facilitates the apoptosis of tumour cells [36].

Thus the aim of the present study was to investigate the effect of quercetin on pro-apoptotic activity of cisplatin in human cervix carcinoma cells (HeLa). The level of MRP, procaspase-3 and Hsp72 were also examined.

Section snippets

Cells and culture conditions

Human cervix carcinoma cell line (HeLa B, ECACC No. 85060701) cultured in RPMI 1640 medium supplemented with 5% FBS (fetal bovine serum) (v/v) was used in this study. Cells were seeded on cover slides (for apoptosis detection and indirect immunofluorescence) or in Falcon vessels (for heat shock proteins, MRP and procaspase-3 identification), at a density of 1 × 105 cells/ml and incubated at 37 °C in humidified atmosphere with 5% CO2.

Drug treatment

Cisplatin (Sigma) at a final concentration of 10 μg/ml and

Quercetin and cisplatin-induced apoptosis of HeLa cells

Quercetin and cisplatin-induced apoptosis was examined by staining HeLa cells with fluorochrome Hoechst 33342 (Table 1). Both quercetin and cisplatin added to the culture medium separately or in combination induced apoptosis. The highest level of apoptosis was observed after 24 h of incubation when flavonoid treatment preceded cisplatin addition and it was time dependant. In 8 h long variant, after preincubation with quercetin 27.33% of apoptotic cells were noticed and it was higher by 7.8% in 24 

Discussion

Since it was discovered that quercetin as a component of human diet, posses several biological activities [24], [46], [47], [48], many studies were performed to understand the mechanisms of its action. Our previous experiments indicated that quercetin inhibited Hsp72 expression in tumour cells and this phenomenon was correlated with increased sensitivity for apoptosis. The flavonoid also appeared as good apoptotic inducer in cells in which Hsp72 expression was blocked by antisense

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