Growth inhibition of combined treatment of cationic liposome/p53 complexes and cisplatin on human carcinoma cells

A combination of cationic liposome/green fluorescence protein (GFP)-p53 complexes and a chemotherapeutic drug, cisplatin, was evaluated for therapeutic activity in human carcinoma cells. Cationic liposome/GFP-p53 complexes and cationic liposome/PEI (polyethylenimine) 0.8k/GFP-p53 complexes were synthesized and evaluated in HeLa and in A549 cells for uptake and cytotoxicity, alone and in combination with cisplatin. The particle size of cationic liposome/GFP-p53 complexes and cationic liposome/PEI 0.8K/GFP-p53 complexes was 318 ± 18 to 754 ± 108 nm, and zeta potentials were -15.7±2.8–+27±08 mV. The GFP expression on the delivery by cationic liposome/pEGFP (enhanced green fluorescence protein) complexes and cationic liposome/PEI 0.8K/pEGFP complexes was demonstrated. Treatment of the cells with either cationic liposome/GFP-p53 complexes or cationic liposome/PEI 0.8K/GFP-p53 complexes inhibited the cell growth. On post treatment with cisplatin, the growth inhibition of the complexes was further increased in HeLa cells and significantly increased in A549 cells on the lipid-to-DNA ratio. This study concluded that the sensitivity to the cancer cells to cisplatin was dependent on the cell line and the ratio of cationic liposome and GFP-p53. GFP-p53 expression delivered by cationic liposome/GFP-p53 complexes would be useful to increase the effect of cisplatin on the treatment of cancer cells.


INTRODUCTION
Cancer is a major disease in the world (Bray et al., 2018). The number of new cases of lung cancer occurred 11.8% and other cancer 53.9%. Lung cancer is the first rank of incidence, mortality, and prevalence by cancer site (WHO, 2018).
Several attempts including p53 gene-based therapy, antisense and siRNA, p53 vaccine, small molecule activating p53, inhibition of p53-mfm2 interaction, and combination of drugs have been tried to activate p53 (Lane et al., 2010). P53 has an important role in the chemotherapeutic drug response of cancer cells. Stabilizing p53, thus increasing in Bax and decreasing in bcl-2, by Vitamin C sensitized cervical carcinoma cells to cisplatin (Reddy et al., 2001). An introduction of p53 cDNA into ovarian cancer cells increased sensitivity to cisplatin (Jin et al., 2002).
Cisplatin has been shown to have efficacy in cancer treatment. A single high-dose cisplatin was more effective than the fractionated doses; however, side effects, such as vomiting and leukopenia still occurred (Li et al., 2019). Synergist treatment has been demonstrated to be an effective alternative to inhibit cancer. Rh-endostatin combined with vinorelbine plus cisplatin improved the survival rate of patients with advanced non-smallcell cancer . A combination of cyclophilin inhibitor and cisplatin has shown synergistic cytotoxicity in human hepatocellular carcinoma cells (Lee, 2010). In addition, a combination of cisplatin and photodynamic therapy has shown a synergistic cytotoxicity in HeLa cells (Wei et al., 2013).
Gene therapy has been depicted to synergize with chemotherapy. Cationic liposome-iNOS gene delivery enhanced the anticancer effect of cisplatin in human lung cancer xenograft mouse models (Ye et al., 2013). Cationic liposome bcl-2 antisensecoated human serum albumin increased the anticancer effect in oral carcinoma cells by the chemotherapeutic drug, doxorubicin (Weecharangsan et al., 2012).
The present study investigated a combined treatment of cationic liposome/p53 complexes and a chemotherapeutic drug, cisplatin on human carcinoma cell growth, HeLa, and A549 cells. This study had a novelty on the combined treatment of cationic liposome/p53 complexes and chemotherapeutic drug on human carcinoma cell growth.

Particle size and zeta potential measurements
Particle size and zeta potential of cationic liposome/ GFP-p53 complexes and cationic liposome/PEI0.8K/GFP-p53 complexes were measured by photon correlation spectroscopy using a Zetasizer (Malvern). The complexes were prepared in DI water, diluted to 1 ml, and measured at 25°C using aqueous flow cell. The amount of cationic liposome/PEI0.8K/GFP-p53 used was 6.25-12.5/1.1/1 µg.

Cell culture
Human cervical carcinoma (HeLa) and human adenocarcinoma (A549) cell lines were used in this study. The cells were grown in MEM with 100 µg/ml streptomycin and 100 U/ml penicillin, 1% amphotericin B, and 10% fetal bovine serum. Cells were incubated in 95% atmosphere and 5% CO 2 at 37°C.

Intracellular delivery and gene expression
pEGFP was transferred into HeLa and A549 cells by cationic liposomes/pEGFP complexes and cationic liposome/ PEI0.8K/pEGFP complexes. Cells were seeded in a 6-well plate at 1.5 × 10 5 cells/well for HeLa and 9 × 10 4 cells/well for A549. After 24 hours, the cells were treated with OptiMEM having cationic liposomes/pEGFP complexes and cationic liposome/PEI0.8K/ pDNA complexes for 4 hours. Cells incubated with pEGFP and without complexes were used as controls. After 4-hour incubation, the cells were rinsed and incubated with the growth media for 24 hours. Following this, cells were rinsed and trypsinized. The number of GFP expressing cells was measured by flow cytometer InCyte software (Guava, Merck, Germany).

Cell growth inhibition
Cell growth inhibition by cationic liposomes/GFP-p53 complexes and cationic liposome/PEI0.8K/GFP-p53 complexes was determined using MTT assay. HeLa cells were cultured in growth media and seeded into 96-well plate (5 × 10 3 cells/well), and let them grown. After 24 hours, the cells were rinsed, and the cationic liposomes/GFP-p53 complexes and cationic liposome/ PEI0.8K/GFP-p53 complexes prepared in 62.5 µl OptiMEM at a weight ratio of 6.25:1 and 6.25:1.1:1, respectively, were transferred to the cells. After 4-hour incubation, the cells were rinsed, growth medium was added, and the cells were further incubated for 24 hours. The cell growth inhibition was measured by MTT assay using a microplate reader (SpectraMax M3, Molecular Devices, San Jose, CA). The cell growth inhibition (%) was calculated by the absorbency of the treated cells relative to the untreated cells.

Chemosensitization of cisplatin to HeLa and A549 cells
Cells were seeded into 96-well plate at 5 × 10 3 cells/well for HeLa cells and 3 × 10 3 cells/well for A549 in growth media. After 24-hour incubation, cells were rinsed and incubated with cationic liposomes/GFP-p53 complexes and cationic liposome/ PEI0.8K/GFP-p53 complexes prepared in 62.5 µl OptiMEM at a weight ratio of 6.25:1 and 6.25:1.1:1, respectively, for 4 hours. After incubation, cells were rinsed and incubated with growth media for 24 hour under 95% atmosphere and 5% CO 2 . After 24-hour incubation, the growth media were replaced with growth media having 4 µM cisplatin and further incubated for 24 hours. The cell growth inhibition was measured by MTT assay as described in cell growth inhibition.

Statistical analysis
The statistical analysis was determined by one-way analysis of variance following with Fisher's Least Significant Difference (LSD) post hoc test. A comparison between means was determined using t-test. The significant level was set at p < 0.05.
DPT liposome/GFP-p53 complexes and DPT liposome/ PEI 0.8 kDa/GFP-p53 complexes were formulated and delivered into HeLa cells and A549 cells. This study showed that DPT liposome and DPT liposome/PEI 0.8 kDa could deliver plasmid DNA into cells. The intracellular delivery and cytotoxicity of the carrier/nucleic acid complexes have been demonstrated to be dependent on the characteristic of carrier and carrier/plasmid DNA ratio and the amount of nucleic acid (Weecharangsan et al., 2017;Zhang et al., 2018) Figure 5 shows the growth inhibition of HeLa cells by cisplatin at a concentration of 0-50 µM. The growth inhibition of HeLa cells increased when the concentration of cisplatin increased from 2.5 to 50 µM. At a cisplatin concentration of 4 µM, growth inhibition of HeLa cells was 8%-10%. Therefore, cisplatin at a concentration of 4 µM was used to evaluate the sensitivity of cancer cells by DPT liposome/PEI 0.8 kDa/GFP-p53 complexes.

Growth inhibition of HeLa cells by DPT liposome/PEI 0.8 kDa/ GFP-p53 complexes and DPT liposome/GFP-p53 complexes
The growth inhibition of HeLa cells by DPT liposome and GFP-p53 was not different from cells treated with a medium. DPT liposome/GFP-p53 complexes significantly inhibited HeLa cell growth (p < 0.05) (Fig. 6). An increasing amount of GFP-p53 from 0.125, 0.25, and 0.5 µg increased the growth inhibition of HeLa cells by DPT liposome/PEI 0.8 kDa/GFP-p53 complexes and significantly at the amount of GFP-p53 of 0.5 µg (p < 0.05) (Fig. 7). Using PEI 0.8 kDa in DPT liposome/PEI 0.8 kDa/ GFP-p53 complexes did not increase the growth inhibition of HeLa cells. The growth inhibition of HeLa cells by DPT liposome/GFP-p53 complexes was higher than that of by DPT liposome/PEI 0.8 kDa/GFP-p53 complexes and by only GFP-p53 (p < 0.05) (Fig. 8).
DPT liposome/GFP-p53 complexes and DPT liposome/ PEI 0.8 kDa/GFP-p53 complexes could inhibit HeLa and A549 cell growth. The growth inhibition of DPT liposome/PEI 0.8 kDa/GFP-p53 complexes was dependent on the concentration of GFP-p53. Song et al. (2012) revealed that cationic lipid-coated PEI/ DNA polyplex increased the transfection efficiency and reduced the cytotoxicity in mesenchymal stem cells. However, effective delivery of plasmid DNA was dependent on the characteristic of PEI and the polymer and plasmid DNA ratio (Costa et al., 2018). This study showed that DPT liposome/PEI 0.8 kDa/GFP-p53   complexes did not show more effective intracellular delivery and gene expression and growth inhibition than DPT liposome/ GFP-p53 complexes in HeLa and A549 cells. In this study, PEI 0.8 kDa resulted in a large particle size of DPT liposome/PEI 0.8 kDa/ GFP-p53 complexes and did not improve intracellular delivery of GFP-p53 from DPT liposome/GFP-p53 complexes, thus not increasing the growth inhibition of cationic liposome/GFP-p53 complexes in HeLa and A549 cells. The particle size of liposome/ PEI/DNA complexes could be dependent on the molecular weight and the structure of PEI.

Growth inhibition of HeLa and A549 cells by DPT liposome/ PEI 0.8 kDa/GFP-p53 complexes in combination with cisplatin
Further treatment with cisplatin, growth inhibition of HeLa cells was slightly increased by DPT liposome/PEI 0.8 kDa/ GFP-p53 complexes at a lipid-to-PEI-to-DNA ratio of 6.25:1.1:1 and DPT liposome/GFP-p53 complexes at a lipid-to-DNA ratio of 6.25:1 (Fig. 9). In A549 cells, growth inhibition was significantly different by DPT liposome/GFP-p53 complexes at lipid-to-DNA ratios of 6.25:1 treated with cisplatin as compared to cells treated with DPT liposome/GFP-p53 complexes at the same lipid-to-DNA ratio and no cisplatin (*p < 0.05) (Fig. 10).
DPT liposome/GFP-p53 complexes and DPT liposome/ PEI 0.8 kDa/GFP-p53 complexes inhibit HeLa cell growth in combination with cisplatin. DPT liposome/GFP-p53 complexes in combination with cisplatin significantly inhibit the A549 cell growth. The combination of DPT liposome/GFP-p53 complexes and cisplatin significantly inhibits A549 cell growth. Liu et al. (2017) depicted that the survival rate of human lung adenocarcinoma cell line was reduced by p53 alpha gene and increased the sensitivity to cisplatin treatment. Ye et al. (2013) showed that cationic liposome/pVAX-iNOS complex enhanced the cisplatin to inhibit cell proliferation of A549 human lung cancer cells and suppression of subcutaneous tumor growth. Liu et al. (2013) demonstrated that cisplatin synergized the TRAIL-induced apoptosis in breast cancer cell lines. Li et al. (2014) showed that the downregulation of p53 by Vasohibin 2 decreased the cisplatin sensitivity to hepatocarcinoma cells.

CONCLUSION
This study concluded that the sensitivity to the cancer cells of cisplatin was based on the cell line and the ratio of cationic liposome and GFP-p53. This study suggests that GFP-p53 expression delivered by cationic liposome/GFP-p53 complexes  GFP-p53 at lipid-to-PEI-to-DNA ratio of 6.25:1.1:1 and DPT liposome/GFP-p53 at lipid-to-DNA ratio of 6.25:1;  = liposomes,  = liposome/GFP-p53 ; *p<0.05 when compared with cells treated with medium, cells treated with GFP-p53, and cells treated with DPT Liposome; # p < 0.05 when compared with untreated cells.  would be useful to increase the effect of cisplatin on the treatment of cancer cells.