Effects of Combination Therapy with Cisplatin and Grape Seed Proanthocyanidins on Human Cervical Cancer Cells C33a

To investigated the inhibitory effect of cisplatin in combination with Grape Seed Proanthocyanidins against human cervical cancer cell lines in vitro. Cervical cancer C33A cells were cultured and divided into four groups: cisplatin group, Grape Seed Proanthocyanidins group, cisplatin with Grape Seed Proanthocyanidins group and the blank control group. Morphologic changes of C33A cells were observed under invert microscope after C33A cells were treated 48, and 72 h. The proliferation of C33A cells was determined by CCK8 assay. Apoptosis were evaluated by PE Annexin V staining. Both of these two agents induced apoptosis and inhibited proliferation in C33A cells in a dose-dependent manner. In combination, cisplatin and Grape Seed Proanthocyanidins amplified these regulatory effects compared to each agent alone. The inhibition of proliferation can be observed in cisplatin group and in Grape Seed Proanthocyanidins group with different concentrations, while the notable effect was showed in Grape Seed Proanthocyanidins group. Combination treatment of cisplatin and Grape Seed Proanthocyanidins produces synergistic influence on C33A cells.


Introduction
Cervical cancer is one of the most common cancers, with an estimated global incidence of 470,000 new cases and over 200,000 deaths per year [1] , making it the second cause of cancer deaths that kills women worldwide. Traditional treatments of cervical cancer include surgery, radiation therapy and chemotherapy.
Among these treatments, chemotherapy combined with operation is the effective one for cervical cancer [2] .
Docetaxe and cisplatin are the most common clinical chemotherapeutic drugs. Cisplatin (DDP) is a platinum Rh metal complex, its center atoms can interfering with DNA replication by binding DNA strands in tumor cells [3] . Cisplatinum is the common clinical platinum antineoplastic, but it has serious cytotoxicity to kidney and bone marrow, this feature restricts its application in cancer treatment [4] . Grape seed procyanidins (GSP), as a kind of polyphenols, exits widely in different kinds of seeds and grape skins. GSP is a kind of free radical scavenger and has strong antioxidation capacity [5] . GSP was proved to regulate cells growth and apoptosis on many kinds of tumor cells cultured in vitro [6][7] .
Therefore, Cisplatin combined with Grape seed proanthocyanidin may enhance their anticancer property and reduce side affect brought by Cisplatin.

Materials
The human cervical cancer cells C33A was obtained from authors' laboratory. DMEM medium was purchased from Hyclone, USA. Fetal bovine serum was from Sijiqing co., LTD. Cisplatin was from sigma.
Grape seed procyanidins was from melonepharma. CCK8 kit was from Bridgen. XDS-1B invert microscope bought from OLYMPUS Company.
Enzyme micro-plate reader bought from Thermo.

Statistical Evaluation
SPSS ver.17.0 software was used for the statistical analysis, and single-factor analysis of variance was used to analyze statistical differences. P < 0.05 was accepted as significant. The data were expressed as the mean ± standard deviation.   From table 2 we conclude that the proliferation inhibition rate GSP onC33A cells for48h was higher than that in control group. The differences had statistical significance(P﹤0.05). From table 3 we conclude that the proliferation inhibition rate DDP&GSP on C33A cells for 48h was higher than that in control group. The differences had statistical significance(P﹤0.05). C33A cell apoptosis rate is 92.3%；

flow cytometry(FCM) detect cell apoptosis rate of C33A
The figure 5 shows that the control group of cell apoptosis rate was 0.1%; the cell apoptosis rate of DDP group which is only add cisplatin for 24h was higher than the control group; the cell apoptosis rate of DDP&GSP group was 92.3%.

Discussion
Cervical cancer is one of the most common gynecological malignancy and 80% of this cases happen in developing countries; there are 529,000 new cases occur each year, and more than half of the patients died. Because of the high cytotoxicity of radiotherapy, chemotherapy drugs, only a patients can suvive over five-year survival afer being treated [8] .
DDP is a chemotherapeutic agent commonly used in the treatment of cervical cancer [9] , and as a cell cycle non-specific drugs, DDP can inhibit the replication of cervical cancer's DNA and has a strong inhabitory effect on kinds of cancers. Research discovered that cervical cancer cells showd lower sensitivity to DDP and producted drug resistance after a period of radiotherapy, increasing the treatment difficulty of cervical cancer.
DDP's toxicity to renel is an important factor in limiting their extensive use. DDP's toxicity to the kidneys is a important factor to limit its extensive use.
The study found that DDP rise renal small tube net stress related proteins GRP78 and P -ERK and endoplasmic reticulum stress response leads to kidney function decline. Grape seed GSP extracted from grape seeds bio-active ingredients and it can increase apoptosis through the mitochondrial pathway protein Bak-1 and reduce apoptotic anti-apoptotic protein Bcl-2-induced cervical cancer cells [10] . GSP in the chemical structure of a phenolic hydroxyl group are easily oxidized quinone structure, which can consume oxygen in the atmosphere, on free radical it has a strong ability to capture and eliminate and the ability to eliminate free radicals in the body is very strong antioxidant. GSP is a good oxygen free radical scavenger. The study found that the appropriate dosage of GSP has certain protective effect on the toxicity of DDP cells and can improve cell survival rate [11] .