Investigation of nedaplatin and CpG oligodeoxynucleotide combination therapy in a mouse model of lung cancer

Purpose: To investigate the anti-tumor effects of nedaplatin (NDP) and CpG oligodeoxynucleotide (CpG-ODN) combination therapy in a mouse-modeled lung cancer. Methods: To evaluate the anti-tumor effects of NDP and CpG-ODN combination therapy, a lung cancer xenograft mouse model was established by subcutaneous injection of LA-795 cells. BALB/c mice were divided into four groups as follows: NDP, CpG-, NDP + CpG-ODN and untreated control group. The sections of lung cancer tissue were stained with hematoxylin and eosin (H&E) and morphologically examined. Spleen, body weight, and spleen index were measured. Flow cytometry was used to determine the proportions of CD3, CD8, CD4 and CD4/CD8 in mice blood cells. Serum levels of interferon-γ (IFN-γ) and interleukin-12 (IL-12) were measured by enzyme-linked immunosorbent assay (ELISA). Results: NDP + CpG-ODN therapy significantly reduced tumor volume and prolonged the survival time of tumor-bearing mice. NDP + CpG-ODN induced a change in cancer cell morphology, including large areas of necrosis which correlated with a reduction in tumor size. NDP + CpG-ODN significantly increased spleen weight/index and dramatically enhanced immune cell activation. This was evident in the increase serum levels of IFN-γ and IL-12. Conclusion: NDP and CpG-ODN combination therapy inhibits the growth of lung cancer and prolongs the survival time of tumor-bearing mice. This may result from the activation of immune cells and increased expression of IFN-γ and IL-12.


INTRODUCTION
Lung cancer is a common malignant cancer with high morbidity and mortality rates.Lung cancer frequently results in organ metastasis, leading to death [1].It is therefore necessary to develop new lung cancer therapy strategies [2].Nedaplatin (NDP) is a platinum-based anti-tumor drug, which is widely used in chemotherapy to treat a variety of cancers.Its prominent antitumor effects are attributed to its interference with DNA replication, resulting in DNA damage [3].However, NDP therapy shows some side effects, including bone marrow suppression, renal dysfunction, and immune dysfunction [4].

Quan & Zhao
Trop J Pharm Res, July 2018; 17(7): 1380 CpG oligodeoxynucleotides (CpG-ODNs), acting as immuno-stimulants, and are composed of single-stranded, non-methylated DNA molecules.Some researchers have demonstrated that CpG-ODN therapy significantly enhances humoral immunity, activating the patient's immune system, and promoting tumor clearance [5].However, the effects of NDP + CpG-ODN combination therapy on lung cancer remains unknown.In order to better understand this relationship we established a lung cancer xenograft mouse model to evaluate the effects of NDP + CpG-ODN combination therapy, as well as, the mechanisms of NDP + CpG-ODN combination therapy.

Animal model
Each mouse was subcutaneously injected with LA-795 cells (10 6 ) in 0.1 mL medium (mouse lung adenocarcinoma cell line, obtained from ATCC, Manassas, VA).When the tumor volume reached 80 mm 3 , sixty mice were randomly divided into 4 groups as follows: control group (no treatment), CpG-ODN group, NDP group, and NDP + CpG-ODN group.All mice received an intravenous injection every two days.In detail, in the control group, the mice received 0.1 mL PBS.In the CpG-ODN group, the mice received 0.1 mL CpG-ODN at a dose of 0.5 mg/mL.In the NDP group, the mice received 0.1 mL NDP (Aladdin Co., Shanghai, China) at a dose of 1 μg/mL.In the NDP + CpG-ODN combination therapy group, the mice received 0.1 mL CpG-ODN at a dose of 0.5 mg/mL and 0.1 mL NDP at a dose of 1 μg/mL .The sequence of nonmethylated CpG-ODN was ATA ATC GAC GTT CAA GCA AG (Shenggong Co., Shanghai, China).After 22 days of treatment, all mice were held without food overnight.The next day the mice were deeply anesthetized with CO 2 and their tissues were isolated.

Therapeutic assessment
The long diameter/length (L) and short diameter/width (W) of the tumor was measured using vernier calipers.According to a previous report [7], tumor volume (V) was calculated as follows: V = (L × W 2 )/2.For the survival experiment, the treatment lasted 22 days.In addition, death of mice was defined by the following conditions: tumor volume > 2000 mm 3 , mouse weight loss > 20 %, or tumor diameter > 20 mm.

Histological examination
Tumor tissues were fixed in 10% formalin before being embedded in paraffin and sectioned (10μm thickness) for histology.The sections were stained with hematoxylin and eosin (H&E) and images were captured by microscopy.

Evaluation of spleen weight and index
The spleens were weighed and measured using the following formula: spleen index = spleen weight/body weight.

Immunocyte assay and enzyme-linked immunosorbent assay (ELISA)
After 22 days of treatment, blood was also collected using a glass capillary tube from the retro-orbital venous plexus under light ether anesthesia.

Statistical analysis
Statistical analyses were conducted using SPSS 11.5 (Chicago, IL, USA).All the data are presented as mean ± standard deviation (SD).Student's t-test and Welch's t-test were used for comparing two groups, while a one-way ANOVA, followed by SNK-q test was used for comparing multiple groups.P < 0.05 was considered statistically significant.

NDP and CpG-ODN therapies attenuate tumor growth in tumor-bearing mice
All treated groups showed slower tumor growth compared to control group.However, from day 10 to day 22, there was a significant increase of tumor volume in the NDP + CpG-ODN combination group, compared to the CpG-ODN group and NDP group.

Morphological changes in by NDP and CpG-ODN therapies
As shown in Figure 3, the histopathological evaluation of tumor tissues showed that the tumor cells in the control group were closely arranged, in round shaped without any apoptotic morphological changes.However, in the treatment groups, the histopathological evaluation of tumor tissues showed obvious vacuoles and nucleus pyknosis.

CpG-ODN or NDP + CpG-ODN combination therapy improve spleen index
As shown in Table 1, the average spleen weight in the CpG-ODN group (0.34 ± 0.07 g) was, significantly higher than controls (0.19 ± 0.03 g).However, there was no significant difference between the NDP group (0.17 ± 0.03 g) and the control group.In addition, the spleen weight in the NDP + CpG-ODN combination therapy group significantly increased compared to the control group and the NDP group.However, there was no significant difference between the NDP + CpG-ODN and CpG group.In addition, the spleen index in both CpG-ODN (20.55 ± 2.62 %) and NDP + CpG-ODN group (20.54 ± 2.65 %) significantly increased compared to controls (11.92 ± 1.87 %).There was no significant difference between NDP and the controls.

Effect of NDP and CpG-ODN therapies on activation of immune cells
As shown in Table 2, the amount of CD4 + , CD3 + and NK cells and the ratio of CD4 + /CD8 + in the CpG-ODN group were significantly increased compared to those in the control group (p < 0.05), whereas the proportion of CD8 + cells was significantly decreased (p< 0.05).However, in the NDP-treated group, the proportion of CD8 + cells was significantly increased, while the CD3 + , CD4 + , NK and ratio of CD4 + /CD8 + significantly decreased compared to those in the control group.In addition, all cells amount in the NDP + CpG-ODN combination therapy group were significantly higher compared to those in the control group (p < 0.05).CpG-ODN, as an immuno-modulator, contains the CpG sequence and effectively activates dendritic cells which secret a variety of immunologic factors [12].In our study, we found an increase in spleen weight in the CpG-ODN treated mice.In addition, the numbers of CD3 + , CD4 + and NK cells, as well as levels of IFN-γ and IL-12 increased in the CpG-ODN group.These findings demonstrated that CpG-ODN enhanced immune function in the tumor-bearing mice.CpG-ODN can activate the NF-κB signaling pathway and induce immuno-stimulation in vivo by binding with toll-like receptor 9 to enter into immune cells [13].Previouse studies also showed that CpG-ODN might induce cytokines, such as IFN-γ and IL-12, by activating JNK1/2 and p38 pathways [14,15].IFN-γ and IL-12 perform functions on activation of T cells by increasing B7-2 and CD69 expression and further promoting the proliferation and differentiation of T cells [16,17].Additionally, CpG-ODN also activates NK cells [14,15].This study investigated the combination therapeutic effects according to the advantage of both NDP and CpG-ODN.The combination of NDP and CpG-ODN showed the significant improvement of the efficiency of lung cancer therapy in animal models.Meanwhile, this combination may also decrease the side effects.We did not present a side effects investigation in this study, but we look forward to studying those mechanisms in the future.

CONCLUSION
The results of this study show that NDP + CpG-ODN inhibits the tumor growth and prolonged the survival time of tumor-bearing mice via activation of immune cells.The therapeutic changes observed indicate that the combination therapy is a potential treatment strategy for the management of lung cancer and reduction in NDP resistance.

Figure 1 :
Figure 1: NDP and + CpG-ODN inhibited tumor growth of lung cancer xenograft mice NDP and CpG-ODN therapies prolong the survival time of tumor-bearing mice As shown in Figure 2, the CpG-ODN (31 days), NDP (35 days) or NDP + CpG-ODN combination (43 days) therapy could significantly prolong the survival time compared to the control groups, which averaged 27 days (p < 0.05).

Figure 2 :
Figure 2: NDP and CpG-ODN therapies prolong the survival time of tumor-bearing mice

Table 1 :
Spleen index and spleen weight of tumorbearing mice (mean ± SD) Compared to the control group, a p < 0.05; compared to the CpG-ODN group, b p < 0.05; compared to the NDP group, c p < 0.05

Table 2 :
Comparison of immune cell populations in tumor-bearing mice in different groups (mean ± SD)
14. Lee S, Hong J, Choi SY, Oh SB, Park K, Kim JS, Karin M, Lee SJ.CpG oligodeoxynucleotides induce expression of proinflammatory cytokines and chemokines in astrocytes: the role of c-Jun N-terminal Quan & Zhao Trop J Pharm Res, July 2018; 17(7): 1384 kinase