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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 12  |  Page : 1035-1040

Norcantharidin combined with diamminedichloroplatinum inhibits tumor growth and cancerometastasis of hepatic carcinoma in murine


1 Institute of Integrated Traditional Chinese Medicine and Western Medicine, Gansu University of Chinese Medicine, Lanzhou, Gansu, China
2 Department of Nursing, Gansu University of Chinese Medicine, Lanzhou, Gansu, China
3 Department of Medical Imageology, Gansu University of Chinese Medicine, Lanzhou, Gansu, China

Date of Web Publication11-Dec-2018

Correspondence Address:
Yong-Qi Liu
Institute of Integrated Traditional Chinese Medicine and Western Medicine, Gansu University of Chinese Medicine, No. 35 Dingxi East Road, Chengguan District, Lanzhou, Gansu 730000
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.192852

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 > Abstract 


Aim: Norcantharidin (NCTD) has been used as a clinical antineoplastic drug in China for several years, and diamminedichloroplatinum is a valuable clinical cancer chemotherapy agent. Here, we tried to investigate the effects of NCTD plus diamminedichloroplatinum on hepatic carcinoma in murine.
Materials and Methods: In vivo and in vitro investigations on anticancer effects of the two drugs were individually made.
Result: In vitro, the combination of the two drugs resulted in apparent apoptosis and cell proliferation inhibitions of H22 cancer cells. Meanwhile, their coadministration in vivo produced significant suppressions of tumor growth and cancerometastasis. Further, CD31 immunohistochemistry and matrigel tube formation assay demonstrated that angiogenesis was inhibited by NCTD plus diamminedichloroplatinum in vivo and in vitro, respectively.
Conclusion: Based on the findings, we concluded that NCTD plus diamminedichloroplatinum may have an additive anticancer efficacy because the two drugs work in different ways, and thus, their combination had inhibited cancer cell proliferations and tumor angiogenesis more effectively than either of the compounds alone.

Keywords: Diamminedichloroplatinum, hepatic carcinoma, norcantharidin


How to cite this article:
Zhang XP, Luo LL, Liu YQ, Liu XS, An FY, Sun SB, Xie XR, Geng GQ, Chen XJ, Li ZD. Norcantharidin combined with diamminedichloroplatinum inhibits tumor growth and cancerometastasis of hepatic carcinoma in murine. J Can Res Ther 2018;14, Suppl S5:1035-40

How to cite this URL:
Zhang XP, Luo LL, Liu YQ, Liu XS, An FY, Sun SB, Xie XR, Geng GQ, Chen XJ, Li ZD. Norcantharidin combined with diamminedichloroplatinum inhibits tumor growth and cancerometastasis of hepatic carcinoma in murine. J Can Res Ther [serial online] 2018 [cited 2019 Oct 17];14:1035-40. Available from: http://www.cancerjournal.net/text.asp?2018/14/12/1035/192852




 > Introduction Top


Mylabris is the dried powdered body of the Chinese blistering beetle. These beetles belong to the family of Moloidae and are widely distributed in China and some parts of the world. Mylabris has been used as a Chinese traditional medicine for more than 2000 years[1] and can produce an anticarcinogenic ingredient cantharidin whose chemical structure is exo-1, 2-syn-dimethyl-3, and 6-oxidohydrophthalic anhydride. However, cantharidin is irritable to the urinary system. In an attempt to reduce the toxicity of cantharidin, its demethylated form, norcantharidin (NCTD) [Figure 1], exo-7-oxabicyclo-(2.2.1) heptane-2, 3-dicarboxylic anhydride, was synthesized by the Diels-Alder reaction with furan and maleic anhydride as the components.[2],[3] NCTD also possesses anticancer activity. It has been used to treat hepatic, gastric, colorectal, and ovarian carcinoma in clinical[3],[4],[5] owing to its multiple anticancer mechanisms: Inducing cell apoptosis, inhibiting the proliferation, blocking tumor invasion and angiogenesis, and so on.[6],[7] Meanwhile, cisplatin (diamminedichloroplatinum [DDP]) plays a crucial role in the treatment of a broad spectrum of malignancies as a valuable chemotherapy agent because it can generate DNA lesions and damage of cancer cells, inhibit the DNA replication of cancer cells.[8],[9],[10] However, the clinical application of large dose chemotherapeutic agents would have serious toxic effects, and the two drugs play anticancer roles though different mechanisms. Therefore, we tried to explore whether a low-dose drugs combination of the two drugs can produce a better curative effect and alleviate toxic effects in this study.
Figure 1: Structures of cantharidin and norcantharidin

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 > Methods Top


Cell culture

Murine hepatic carcinoma cell line H22 was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C with 5% CO2 in the atmosphere.

Tumor models

Six-week-aged female BALB/c mice were challenged with 3 × 105 tumor cells s.c. in the right flank. According to the previous literatures,[11],[12] when the tumors grew to ~200 mm3, the mice were injected intraperitoneally once every 3 days per mouse per injection with: (1) NS group: Normal saline; (2) DDP group: 5 mg/kg DDP; (3) NCTD group: 3 mg/kg NCTD; (4) combined treatment group: 2.5 mg/kg DDP and 1.5 mg/kg NCTD.

Tumor dimensions were measured every 3 days with calipers, and tumor volumes were calculated according to the following formula:

Width2 × length × 0.52.

In addition, to investigate the effect of the drugs on cancer cells metastasis, pulmonary cancerometastasis models of H22 cancer cells were established. Briefly, BALB/c mice were injected with 3 × 104 cells intravenous, and the treatments were administered after 24 h of injections. After 2 weeks, the mice were sacrificed and pulmonary cancerometastasis nodules were counted.

CD31

Specific staining for endothelial cells was conducted using a monoclonal rabbit anti-mouse immunoglobulin. Slides were fixed in cold acetone for 20 min. After two washes with phosphate buffer saline, the sections were treated with 3% hydrogen peroxide to inactivate endogenous peroxidase. After washing, they were blocked with rabbit sera at 37°C for 15 min, incubated with primary antibodies of CD31 at 37°C for 2 h, incubated with secondary antibody for 40 min, and then tertiary antibodies for 40 min. Diaminobenzidine was added as a chromogen. Tissue sections were stained under a microscope, rinsed in water, and restained with hematoxylin. Vessel density was determined by counting the number of microvessels per high-power field in the sections.

Endothelial cell capillary-like tube formation assay

In vitro matrigel tube formation assay was performed to determine the effect of the two drugs on angiogenesis as previously described.[13] Briefly, Mouse vascular endothelial cells MS1 (8 × 104 cells) were seeded onto matrigel-coated 96-well plate with NCTD (2 μg/ml), DDP (2 μg/ml), or NCTD (1 μg/ml) plus DDP (1 μg/ml) in the cell culture medium. Tubular structures in the matrigel were examined under microscope at ×100 magnification after 72 h incubation.

Cell viability assay

H22 cells were incubated in 96-well plates at a density of 3 × 104 cells per well. After treatment for 48 h with NCTD (5 μg/ml), DDP (5 μg/ml), or NCTD (2.5 μg/ml) plus DDP (2.5 μg/ml), the cell viability of the treated samples was assessed using the 3-(4, 5-dimethylthiazol-2-yl)-5-diphenyltetrazolium bromide (Sigma) assay, as described previously.[14] The absorbance was measured at a wavelength of 570 nm. The cell proliferation rate was calculated according to the absorbance value, inhibition rate of cell proliferation = (absorbance value of control group − absorbance value of experimental group/absorbance value of control group) ×100%.

Cell apoptosis assay

Briefly, after 24 h treated with different drugs, Cells were incubated with annexin VFITC and PI for 10 min in the dark at 25°C according to the manufacturer's guidelines, before suspension in binding buffer. Cell apoptosis was analyzed by an FACScan flow cytometer (Becton Dickinson, San Jose, CA).


 > Results Top


Effect of norcantharidin or/and diamminedichloroplatinum on cell proliferations and apoptosis

H22 cells in logarithmic growth phase (24-well plates at 1 × 105 cells/well) were separately exposed to NCTD (5 μg/ml), DDP (5 μg/ml), and NCTD (2.5 μg/ml) plus DDP (2.5 μg/ml). After 48 h, visible inhibitions on cell proliferations occurred in DDP group (27.7 ± 1.21%) and NCTD group (20.9 ± 1.46%), and the most significant inhibition was observed in NCTD plus DDP group (41.8 ± 1.02%) (P < 0.01) [Figure 2]. Furthermore, NCTD plus DDP induced the remarkable apoptosis of cancer cells (P < 0.05) [Figure 3].
Figure 2: Effect of norcantharidin or/and diamminedichloroplatinum on cell proliferations. Respectively, H22 cells were exposed to: (a) Normal saline, (b) 5 μg/ml norcantharidin, (c) 5 μg/ml diamminedichloroplatinum, (d) 2.5 μg/ml norcantharidin plus 2.5 μg/ml diamminedichloroplatinum. Norcantharidin plus diamminedichloroplatinum produced the most visible suppression on cancer cell proliferations (P < 0.01)

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Figure 3: Apoptosis of cancer cells. Cancer cells were treated, respectively, with norcantharidin (5 μg/ml), diamminedichloroplatinum (5 μg/ml), and norcantharidin (2.5 μg/ml) plus diamminedichloroplatinum (2.5 μg/ml) for 48 h. Cell apoptosis was detected by FACScan flow cytometer. The remarkable apoptosis occurred in norcantharidin plus diamminedichloroplatinum group (P < 0.05)

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Effect of norcantharidin or/and diamminedichloroplatinum on tumor growth

The therapeutic efficacy of NCTD or/and DDP was tested in the established tumors. The treatment was administrated on day 7 after the subcutaneous injection of H22 hepatic carcinoma cells when the tumor was perceptible by touch. Mice were intraperitoneally injected once every 3 days with NS, NCTD (3 mg/kg), DDP (5 mg/kg), and NCTD (1.5 mg/kg) plus DDP (2.5 mg/kg), respectively. NCTD plus DDP exhibited a significant inhibition of tumor growth (P < 0.05) [Figure 4].
Figure 4: Inhibition of tumor growth. Tumor-bearing mice were injected subcutaneously with normal saline (◊), 3 mg/kg norcantharidin (Δ), 5 mg/kg diamminedichloroplatinum (◼), 1.5 mg/kg norcantharidin plus 2.5 mg/kg diamminedichloroplatinum (×), respectively. There was a significant difference in tumor volumes between combination therapy group and other groups (P < 0.05) (n = 10)

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Effect of norcantharidin or/and diamminedichloroplatinum on cancer metastasis

In the model of pulmonary cancerometastasis, use of NCTD or DDP alone demonstrated mild suppressions on cancerometastasis. Although NCTD plus DDP did not eradicate the metastasis of cancer cells completely, fewer lung surface nodules illustrated that combined treatment had an efficient block on cancerometastasis [Figure 5]b. Moreover, hematoxylin and eosin staining confirmed the results by the decline of metastatic colonies [Figure 5]c in the section.
Figure 5: Effect on lung metastasis of cancer cells. BALB/c mouse lung metastasis model was established by intravenous injection with 3 × 105 H22 cells. Mice were sacrificed 14 days after tumor cell inoculation. (A) Representative lungs, (B) number of lung surface nodules, (C) hematoxylin and eosin staining of lung tissues in H22 metastasis model: (a) Normal saline, (b) diamminedichloroplatinum, (c) norcantharidin, (d) norcantharidin plus diamminedichloroplatinum. The lungs from norcantharidin plus diamminedichloroplatinum mice showed less lung surface metastasis nodules (P < 0.05) (n = 10)

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Effect of norcantharidin or/and diamminedichloroplatinum on angiogenesis

To investigate the effect of NCTD or/and DDP on angiogenesis within tumors, immunohistochemical staining of frozen sections was administrated with antibodies against CD31 and angiogenesis within tumor tissue was estimated by counting the number of microvessels in the sections. In comparison with NS group, microvessel densities in both NCTD and DDP groups were reduced, but the most apparently decrease was found in NCTD plus DDP group (P < 0.05) [Figure 6]. The suppressing effect was further verified by matrigel tube formation assay of mouse endothelial cell MS1in vitro[Figure 7]. The tube formation rate was 39.3%, 41.6%, and 21.3% in NCTD (2 μg/ml), DDP (2 μg/ml), and NCTD (1 μg/ml) plus DDP (1 μg/ml) group, respectively [Figure 7].
Figure 6: Inhibition of angiogenesis within tumors. CD31 immunohistochemistry showed the significant inhibition of angiogenesis within tumors by norcantharidin plus diamminedichloroplatinum (P < 0.05), (a) Normal saline, (b) diamminedichloroplatinum, (c) norcantharidin, (d) diamminedichloroplatinum plus norcantharidin, (e) microvessel density determined by counting the number of microvessels per high-power field in the sections stained with antibodies reactive to CD31 (n = 10)

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Figure 7: Matrigel tube formation assay of mouse vascular endothelial cells MS1. After treated, respectively, with norcantharidin (2 μg/ml), diamminedichloroplatinum (2 μg/ml), and norcantharidin (1 μg/ml) plus diamminedichloroplatinum (1 μg/ml) for 72 h, and tubular structure in each group was quantified by manual counting. Norcantharidin plus diamminedichloroplatinum showed obvious inhibition on tube formation (P < 0.01). (a) Normal saline, (b) diamminedichloroplatinum, (c) norcantharidin, (d) diamminedichloroplatinum plus norcantharidin (n = 10)

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Toxicity observation

In the treatment period, no visible adverse effect was observed in gross measures such as ruffling of fur, weight loss, behavior, and life span. Furthermore, no histopathology changes in the liver, spleen, kidney, lung, and heart were found by microscopic examination.


 > Discussion Top


DDP is mostly used for a variety of tumors as a chemotherapeutic agent. Mainly, it interacts with DNA to produce DNA crosslink adducts, which can activate some signal transduction pathways, involving p53, p73, mitogen-activated protein kinases, and ATM-Rad3-related protein kinase, followed by the activation of cancer cells apoptosis.[15],[16] While the antitumor activities of NCTD are multifarious: It can lead to apoptosis, inhibit tumor angiogenesis and cancerometastasis, and effect multiple pathways controlling cell proliferations.[6],[7],[17],[18] More importantly, unlike some other conventional chemotherapeutics, NCTD is preferentially toxic to cancer cells rather than normal cells.[19] Our results showed that NCTD plus DDP had inhibited proliferations of cancer cells [Figure 2], induced significant apoptosis in vitro[Figure 3], and suppressed tumor growth in vivo[Figure 4]. These results indicated that NCTD plus DDP had surely a better therapeutic action. It is possible that the two drugs' antitumor effects were additive.

Angiogenesis is crucial for tumor growth and cancer progression,[20],[21],[22],[23] and tumor microvessel helps tumor to get its metabolic requirements.[23],[24],[25] Previous studies found that NCTD inhibits tumor angiogenesis by blocking vascular endothelial growth factor receptor 2/(mitogen-activated protein kinase) signaling pathways,[26],[27] and DDP exerts its antiangiogenic ability by inhibiting the proliferations of blood vessel endothelial cells.[28] Here, NCTD and DDP showed different degrees of inhibitory effect on angiogenesis in vivo[Figure 6] and in vitro[Figure 7], and the most visible inhibition was caused by their combination. It may be that DDP can strengthen the antiangiogenesis efficacy of NCTD due to its proliferation inhibition on vascular endothelial cells. More, metastases of cancer cells are dependent on neovascularization.[22],[23],[24],[25],[29] In our study, fewer pulmonary metastasis nodules [Figure 5]a and metastatic colonies [Figure 5]c raised the possibility that antiangiogenesis by the combination treatment had also caused the block of cancer cells metastasis.

Based on the findings, the reasons why the coadministration of the two drugs can improve therapeutic effect could lie in two aspects: The inhibitions of cancer cells proliferations and the suppressions of tumor angiogenesis. NCTD-DDP combination could have an additive anticancer efficacy because the two drugs work in different ways.

Financial support and sponsorship

The study was supported by the talent introduction program in Gansu University of Chinese Medicine, the National Natural Science Foundation of China (Grant No. 81360345) and Natural Science Foundation of Gansu Science and Technology Department (Grant No. 1208RJZA221).

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]



 

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