The use of mitochondrial targeting resveratrol liposomes modified with a dequalinium polyethylene glycol-distearoylphosphatidyl ethanolamine conjugate to induce apoptosis in resistant lung cancer cells
Introduction
The multidrug resistance (MDR) of cancers is a major obstacle to successful cancer chemotherapy, and it results in incomplete therapeutic response, recurrent and metastasis of cancers [1]. Intrinsic MDR plays a crucial role in the drug resistance and is caused by genetic and epigenetic changes of cancer cells through altering function of pro-apoptotic or apoptotic genes encoded proteins such as Bcl-2 family proteins [2], [3] and capase proteins involved in the apoptosis signaling pathway [4], [5].
There are two basic ways to eliminate cancer cells in the chemotherapy. One approach is to kill cancer cells by direct exposure of cancer cells to toxic chemicals. The other one is to induce the suicide of cancer cells, namely, induction of apoptosis which has been regarded as a very important strategy for thoroughly eliminating tumors [6].
Mitochondria are membrane-enclosed organelles found in most eukaryotic cells, and are described as "cellular power plants". Besides supplying cellular energy, mitochondria are involved in a range of other processes, such as cellular differentiation, cell death, as well as the control of the cell cycle and cell growth [7]. Cancer cell mitochondria are structurally and functionally different from their normal counterparts [8]. They exhibit an extensive metabolic and are more susceptible to mitochondrial perturbation than normal cell mitochondria [9].
Cancer cell apoptosis induced by anticancer drugs may involve two independent initiator pathways that lie in upstream of all these effector events: activation of upstream caspase by cross-linking with death receptors on their ligands, and the release of apoptogenic factors by triggering various forms of cellular stress [10]. Both of them seem to be focused on the mitochondrial level, thereby triggering the cell apoptosis. Consequently, induction of apoptosis through direct targeting of mitochondria of cancer cells could be a strategy to circumvent the intrinsic drug resistance in chemotherapy [4]. Based on the above premises, mitochondria-targeting therapy emerges as a promising means to selectively eliminate cancers [11].
A number of experimental drugs have been explored to act primarily on mitochondria to induce apoptosis of cancer cells, such as glucose transporter inhibitors [12], mitochondrial permeability transition (MPT) modulators [13], [14], mitochondrial outer membrane permeabilization (MOMP) modulators [11], and so on. However, these agents are less than ideal due to unfavorable pharmacokinetic property which leads to extensively systemic distribution in the body, hence increasing the systemic toxicity of the agents.
To date, many mitochondrial drug delivery systems have been constructed to solve these problems. For examples, triphenylphosphonium cation (TPP), dequalinium (DQA) and mitochondrial targeting signal peptides (MTSs) have been packed or modified onto the lipid carriers such as mitochondriotropic molecules for targeting mitochondria [15].
Resveratrol is a polyphenol and is widely distributed in plant foods such as grapes wine, and peanuts. Resveratrol has emerged as a potential antitumor agent having the ability to inhibit all three major stages of carcinogenesis, including initiation, promotion and progression [16]. In variety types of cancer cells, resveratrol can stimulate apoptosis through activating the mitochondrial apoptotic pathway [17], [18], [19]. However, the low water solubility, stability and therapeutic index of resveratrol make it unsuccessful in the clinical therapy [20].
We proposed here a strategy that mitochondrial targeting resveratrol liposomes could be able to trigger apoptosis by acting on mitochondria of cancer cells, and be used a co-therapy agent with a cytotoxic drug for overcoming the intrinsic resistant cancers.
In the present study, we synthesized a targeting material, dequlinium-polyethylene glycol distearoylphosphatidylethanolamine (DQA-PEG2000-DSPE), which was used as mitochondriotropic molecule for modifying the surface of liposomes. The objectives of the present study were to characterize the mitochondrial targeting resveratrol liposomes, to evaluate their potential apoptosis inducing effect on the resistant lung cancers, and to assess their therapeutic efficacy in combination with a cytotoxic formulation, vinorelbine liposomes.
Section snippets
Materials
Dequalinium (DQA) was purchased from Hangzhou Sanhe Chemicals Co., Ltd. (Hangzhou, China). 1,2-distearoyl-sn-glycero-3-phosphoethamolamine-N-[carboxy (polyethylene glycol) 2000] (COOH-PEG2000-DSPE) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). 1-hydroxy-1H-benzotriazole (HOBt) was purchased from Sigma–Aldrich (St. Louis, MO, USA). Polyethylene glycol-distearoylphosphatidylethanolamine (PEG2000-DSPE) was purchased from NOF Corporation (Japan). Resveratrol was obtained from
Synthesis and characterization of DQA-PEG2000-DSPE
Fig. 1 shows the synthetic scheme for DQA-PEG2000-DSPE. To synthesize DQA-PEG2000-DSPE conjugate, DQA was conjugated through coupling one of the aromatic amino groups of DQA to the carboxylic group on COOH-PEG2000-DSPE, using DCC and HOBt as coupling agents.
Fig. 2 shows the MALDI-TOF-MS spectra of dequalinium (DQA), DQA-PEG2000-DSPE conjugate, and the uncoupled COOH-PEG2000-DSPE. The MALDI-TOF spectra of production after conjugating reaction exhibited the average mass of DQA-PEG2000-DSPE at m/z
Discussion
In the present study, a DQA-PEG2000-DSPE conjugate is first synthesized, and modified onto the mitochondrial targeting resveratrol liposomes, which show apoptosis inducing effect on the resistant lung cancer cells by targeting mitochondria of cancer cells. DQA is a quaternary ammonium cation commonly available as the dichloride salt, and an amphiphile with delocalized cationic charge centers showing selectively mitochondrial accumulation [29]. DQA has been used as mitochondrion-tropic molecule
Conclusions
A DQA-PEG2000-DSPE conjugate was synthesized by acrylation from dequalinium and COOH-PEG2000-DSPE with a yield o 87%, and could be used as mitochondriotropic molecule. The mitochondrial targeting liposomes are able to punctate accumulated in mitochondria of resistant A549/cDDP cells when encapsulating coumarin as the fluorescent probe. Furthermore, mitochondrial targeting resveratrol liposomes exhibit significant antitumor efficacy in resistant A549/cDDP cells by inducing apoptosis through
Acknowledgements
This paper was supported by the Key Grant of Beijing Natural Science Foundation (No. 7091005), and by the National Basic Research Program of China (973 program, 2007CB935801)
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