Background
Genes are transcribed into mRNAs and subsequently translated into proteins to carry out the major functions within a cell and the mutations in certain genes leading to their suppression or overexpression are usually responsible for both acquired and genetic diseases. Delivery of functional gene(s) or gene-silencing element(s) could be the potential options in restoring the normal functions of the cell. RNA interference (RNAi) that can selectively silence mRNA expression in cell cytoplasm can be utilized to develop new drugs against target therapeutic genes [
1‐
5]. RNAi can be harnessed for selective gene inhibition in two different routes: 1) cytoplasmic delivery of short interfering RNA (siRNA) for directly breaking down the specific mRNA and 2) nuclear delivery of gene expression cassettes to express a short hairpin RNA (shRNA) which is further processed by cellular machinery to siRNA in the cytoplasm [
6]. However, siRNA, a synthetic RNA duplex of 21–23 nucleotides, is more advantageous than shRNA because of the difficulty in the construction of a shRNA expression system [
6], and the requirement of the expression system to overcome the nuclear barrier for shRNA expression [
7]. siRNA in the cytoplasm of the cells incorporates into a multiprotein RNA-induced silencing complex (RISC) and is unwound into single-stranded RNAs by Argonaute 2, a multifunctional protein within the RISC, forming antisense strand-associated RISC in order to guide and selectively degrade the complementary mRNA with the help of Argonaute-2 [
8]. Perfect hybridization between the antisense strand of siRNA and the target mRNA leads to degradation of the mRNA near the center of the target-siRNA duplex [
8]. However, the strong anionic phosphate backbone with consequential electrostatic repulsion from the anionic cell membrane is an obstacle to the passive diffusion of siRNA across the membrane [
9]. The hydrophobic lipid bilayer could pose an additional barrier to the hydrophilic siRNA. Moreover, naked siRNA can be degraded by the plasma nucleases and even subjected to renal elimination due to its small size before reaching the target site
in vivo[
10,
11]. A number of existing non-viral vectors have been developed for intracellular siRNA delivery with limited efficacy [
8]. Usually, a non-viral vector being cationic can electrostatically bind with an anionic siRNA to form a stable complex, thus protecting it from nuclease-mediated degradation, enabling it to cross the plasma membrane through endocytosis and finally facilitating its endosomal escape [
8].
Cancer is a complex disease responsible for millions of deaths worldwide and despite remarkable efforts made in the last decades limited successes have been achieved so far to cure various types of cancer. Clinical efficacy of current chemotherapeutic drugs are often limited owing to to their toxic effects on normal cells and the patients can tolerate only the doses which are therapeutically insufficient, thus leading to chemoresistance and subsequent tumor recurrence [
12]. Since cancer is the result of overexpression or suppression of signaling pathways aiding cancer cell survival and proliferation, non-viral vector-mediated delivery of siRNAs specific for the genes of pathways, to cancer cells would be the potential treatment options that might additionally render cancer cells extremely sensitive to cytotoxic chemotherapy [
11]. Among the signalling cascades, MAP kinase, PI-3 knase and Ca
2+-calmodulin pathways are extensively involved in proliferation and survival of various cancer cells [
13‐
15]. On the other hand, conventionally used chemotherapy drugs induce apoptosis of cancer cells by interfering with the major cellular functions which might have some of cross-talk with the components of cell proliferation/survival pathways. siRNA-mediated knock-down of the genes encoding the enzymes of those pathways, therefore, might not only slow down the growth of cancer cells, but also sensitize them to anti-cancer drugs.
In Ca
2+-calmodulin pathway, stimulation with growth factor either G protein-coupled receptors or receptor tyrosine kinases activates the phospholipase C (PLC) enzyme, which, in turn, hydrolyses the membrane phospholipid, phosphatidylinositol 4, 5 bisphosphate (PIP2) to diacylglycerol (DAG) and inositol (1,4,5) trisphosphate (IP3). DAG activates PKC while IP3 binds to its receptor on the endoplasmic reticulum allowing diffusion of Ca
2+ from the ER to increase intracellular [Ca
2+[
16]. The released Ca
2+ binds to calmodulin (CaM) and Ca
2+/CaM functions as an allosteric activator of a considerable number of protein kinases regulating cell proliferation and apoptosis [
17].
Recently, we have developed an efficient siRNA delivery system based on some unique properties of carbonate apatite- electrostatic affinity for binding anionic siRNA, ability of preventing crystal growth for generation of nano-size particles for efficient endocytosis and fast dissolution kinetics in endosomal acidic compartments to facilitate the release of siRNA from the particles as well as from the endosomes, leading to the efficient silencing of reporter gene expression. Moreover, nanoparticle-assisted delivery of validated siRNA against cyclin B1 resulted in the significant inhibition of cancer cell growth [
18,
19].
Here we show that carbonate apatite-mediated delivery of siRNA against PLC-gamma-2 (PLCG2) and calmodulin 1 (CALM1) genes sensitized a human cervical cancer cell line (HeLa cell) to doxorubicin- and paclitaxel-induced cell death depending on the doses of the drugs while no such synergistic effect was observed with cisplatin, another commonly used chemotherapy drugs.
Methods
Reagents
MTT (3-(4,5-Dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide) and DMEM were purchased from Molecular Probes, Sigma and Gibco BRL, respectively. Validated siRNAs against PLCG2 (Target sequenc 5'-GACGACGGTTGTGAATGATAA-3') and CALM1(Target sequence 5'-CGGCAACTTACACACATTGAA-3') were obtained from Qiagen. Upon delivery in the lyophilized form, the siRNAs were diluted to obtain a 20 μM solution using RNAse-free water and allocated into multiple reaction tubes for storage at -20o C as repeated thawing might affect siRNA’s silencing efficiency.
Cell culture
HeLa cells were cultured in 25-cm2 flasks in Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL) supplemented with 10% fetal bovine serum (FBS), 50 μg penicillin ml-1, 50 μg streptomycin ml-1 and 100 μg neomycin ml-1 at 37 °C in a humidified 5% CO2-containing atmosphere.
Cells from the exponentially growth phase were seeded at 50,000 cells per well into 24-well plates the day before transfection. 3 μl of 1 M CaCl
2 was mixed with 10 nM of siRNA in 1 ml of fresh serum-free HCO
3- (44 mM)-buffered DMEM medium (pH 7.5), followed by incubation at 37 °C for 30 min for complete generation of siRNA/carbonate apatite particles [
18,
19]. 10% FBS and (depending on the experimental conditions) 0.2 to 1 μM drugs (cisplatin, doxorubicin, paclitaxel) had been mixed with the medium containing the siRNA/apatite complexes before the medium was added onto the rinsed cells. The cells were subsequently cultured for 48 h prior to the assessment on cell viability [
18,
19].
Cell viability assessment with MTT assay
30 μl of MTT solution (5 mg/ml) was added onto the cells in each well of the 24-well plate and incubated for 4 hr at 37 °C. 0.5 ml of DMSO was added after removal of the medium from each well to resolve the crystals, followed by incubation for 5 min at 37 °C. Absorbance was measured in a micro plate reader at 570 nm with a reference wavelength of 630 nm. Each experiment was done in triplicate with the data representing mean value ± SE (n = 3) and being statistically significant (< 0.05).
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
AS has originally planned the project in close discussion with EHC and TA and finally carried out the experiments in collaborations with MJC, APK and SH. All authors read and approved the final manuscript.