With the continuous development of ovarian cancer technology, the prognosis of patients has been improved, while the mortality rate is still the first in gynecological cancer[21], especially for the late recurrence of OC patients, the survival rate has not been ideal. More importantly, the widespread use of chemotherapy drugs, damaged the patients’ normal tissues, and even drug-resistance, leading to the failure of treatment[22]. The problem may be solved by finding drugs that more effective or can reverse drug-resistance, or reducing the dosage of chemotherapy drugs without affecting the therapeutic effect. In our initial experiment, SKOV-3/DDP was used as the research object to preliminarily explore the synergistic effect of targeting drug Ap combined with chemotherapy drug nab-P on OC. Our results showed that AP and nab-P all have strong antitumor effects on SKOV-3/DDP, and when they are combined, their respective antitumor effects were enhanced. Furthermore, when the two drugs combined, if nab-P properly reduced, the two drugs can still achieve ideal antitumor effect. This means that if consistent with the results of clinical trials, such a drug scheme may possibly be used to reduce the adverse reactions caused by chemotherapy drugs. That’s why we designed this paper.
Firstly, we obtained the IC-50 value of SKOV-3/DDP to nab-P and AP. The results showed that IC-50 of nab-P was lower than AP, this indicated that the drug effect of nab-P on SKOV-3/DDP is slightly stronger than that of AP. Apoptosis is a form of cell death after the initiation of suicide procedure, which plays an important role in maintaining the balance between normal cell death and cell division[23]. Inhibition of apoptosis and abnormal cell proliferation and differentiation are the biological basis of tumorigenesis[24]. Promoting apoptosis of tumor cells is one of the most important methods in tumor targeted therapy[25]. The Bax and bcl-2 are the key to regulate apoptosis. So in our study, Western blot and Immunofluorescence were applied to analyze the expression of apoptosis related protein Bax, bcl-2, the results showed after nab-P and AP intervention, Bax was higher than control group, bcl-2 was contrary. That’s means nab-P and AP all can play the satisfactory anti-tumor effects on SKOV-3/DDP, which can promote tumor cell apoptosis. Tumor growth mainly depends on tumor angiogenesis[26]. Vascular endothelial growth factor(hereinafter referred to as VEGF) is an important factor in angiogenesis, it widely distribute in various normal tissues and tumor tissues, can stimulate the proliferation, survival and migration of endothelial cells through the corresponding cell surface receptors and signal pathways, which is one of the important factors for the growth and survival of endothelial cells[27]. VEGFR is the main functional receptor of VEGF, and VEGFR-2 is considered to be the key molecule of VEGF signaling pathway to induce angiogenesis. So, it’s an effective way to treat tumor by inhibiting angiogenesis[28]. Apatinib is the ATP binding site of VEGFR-2, which can inhibit the production of p-VEGFR-2 and block the downstream signal transduction pathway. Therefore, we tested expression of p-VEGFR-2 in different groups. The results showed that in AP group, the expression of p-VEGFR-2 was reduction, while in nab-P group was slightly higher than that in Control group. In the way of MMP-2, previous studies have shown that matrix metalloproteinases plays an important role in tumor cell invasion, the invasion and migration is the main cause of tumor metastasis[29] and MMP-2 is an important part[30]. In experience groups, the expression of MMP-2 were declined when compared with Control group. We also used Transwell and Cell scratch experiments to verify the changes of invasion and migration of SKOV-3/DDP in different groups. Which was the same as the expected results, after the effects of nab-P and AP, the number of invasive cells decreased significantly, and the 24-hour cell healing rate decreased. Therefore, we can draw the first conclusion that nab-P and AP can play a good anti-tumor effect on drug-resistant SKOV-3/DDP. This results are the same with Jing, Momeny, Tipton et al[31–33].
However, considering the limitation of single drug therapy, combined drugs is a routine means for clinical tumor treatment[34], for the treatment of ovarian cancer, the combination of chemotherapy targeted drugs has made a great breakthrough in the basic research and clinical cases in recent years[19, 35–36]. The nab-P is a kind of paclitaxel wrapped by human serum albumin, which can enter the tumor cells easily, increase the drug concentration in tumor cells, and achieve ideal therapeutic effect on drug-resistant ovarian cancer. However, after the therapy of nab-P, the decrease of tumor load may increase the expression of VEGF signal[37]. Therefore, if VEGFR-2 inhibitor is used at the same time, the expression of VEGFR-2 will be continuously inhibited, and with VEGF disabled,tumor angigogenesis will be reduced simultaneously, so as to achieve better antitumor effect. Fortunately, AP is a small molecule targeted drug to inhibit VEGFR-2, so we look forward to the better performance of combination of these two drugs.. According to this problem, we have carried out the next cytological experiments. We also tested the inhibitory effect of nab-P combined Ap on SKOV-3/DDP by MTT assay, and with the help of compusyn (version 2.0) software analysised the combination index of the two drugs, the results showed that nab-P combined with AP had a strong inhibitory effect on SKOV-3/DDP, and when they work together, they worked synergism effect. According to the IC-50 value, we set up the combination of nab-P with different concentration gradients combined with AP, Western blot and immunofluorescence experiments also be used to verify the expression of related proteins(Bax, bcl-2, p-VEGFR-2 and MMP-2), the results showed that when nab-P combined with AP, bcl-2, p-VEGFR-2 and MMP-2 were all expressed higher than Control group(P < 0.01), while Bax was lower, and in this three experience groups, there were no significant difference in this protein’s expression. Transwell and Cell scratch test’s results also showed the combination of drugs greatly weakened the invasion and migration of SKOV-3/DDP. Above that, we can obtain another conclusion that nab-P combined with AP can promote the apoptosis and inhibit the invasion of SKOV-3/DDP. When nab-P was reduced at a suitable concentration, the anti-tumor effect of combined AP has little effect. We can think that at the cellular level, when nab-P and AP are combined, the drug toxicity can be reduced by reducing the dosage of nab-P.
After obtaining satisfactory results at the cell level, we designed animal experiments to verify whether the results at the animal level are consistent with those at the cell. Different concentration gradients of nab-P were set up with the same combination of AP. The reduction of tumor mass were observed by measuring the volume of subcutaneous tumor in nude mice after administration, the results showed that after the treatment of nab-P combined with AP, the tumor volume decreased significantly, and the tumor inhibition rates in the experimental group were 90.08%(Group-1), 88.7%(Group-2) and 88.4%(Group-3), respectively. There was no significant difference among the three experimental groups, that is to say, after combination of AP, reduction of nab-P had little effect on anti-tumor. In addition, Western blot, immunofluorescence and immunohistochemistry were carried out to detect Bax, bcl-2, CD31, p-VEGFR-2 and MMP-2. The results showed that the expression of apoptosis related protein bcl-2, vascular related protein CD31, p-VEGFR-2 and invasion related protein MMP-2 decreased significantly and Bax increased, compared with the control group, the difference was statistically significant, but there were no obvious difference among the three groups, which was highly consistent with the experimental results at the cell level. The side effects caused by chemotherapy drugs while killing tumor cells are important factors affecting the quality of life of patients[38]. In order to verify the effect of reducing the dosage of nab-P on the survival of nude mice, we set up two observation indexes: daily average diet and one hour exercise. We found that there was a significant negative correlation between the quality of life of mice and the intervention of nab-P. Therefore, we can draw the third conclusion, that is, in vivo experiments, after nab-p combined with AP, appropriate reduction of the recommended dosage of nab-p can achieve the purpose of reducing the adverse reactions caused by nab-P in mice.
In a word, in this experiment, we combined the targeting drug Apatinib with the chemotherapy drug paclitaxel (aluminum binding type) to act on the human ovarian cancer cell line and xenograft tumor model, and found that the combination of the two drugs can significantly inhibit the proliferation and invasion of the tumor. Secondly, after the combination, it can be properly reduced the inhibitory effect of low nab-P dose on OC cell line and xenograft tumor model. This suggests that, in the clinical treatment, the combination scheme can bring less side effects. Due to the certain toxicity of chemotherapy drugs, patients are prone to some adverse reactions such as myelosuppression, gastrointestinal reactions, etc. During the chemotherapy period, some patients are showed well intolerable, and by reducing the dose of chemotherapy drugs, it will lead to a great discount in the treatment effect. Therefore, if the combination can reduce the dosage of nab-P on the basis of not reducing the therapeutic effect, and the adverse reactions will be correspondingly reduced, which may improve the quality of life of patients.
Based on the above all, the combination of nab-P and Ap performed well in inhibiting the growth of tumor, and improving the drug efficacy and the survival rate of patients. In addition, it can also reduce the drug dose of nab-P, thereby reducing the adverse reactions brought by nab-P to improve the quality of life of patients.