Journal of Cancer Research and Therapeutics

: 2018  |  Volume : 14  |  Issue : 12  |  Page : 1035--1040

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

Xiao-Ping Zhang1, Lu-Lu Luo2, Yong-Qi Liu1, Xue-Song Liu1, Fang-Yu An1, Shao-Bo Sun1, Xiao-Rong Xie1, Guang-Qin Geng1, Xue-Juan Chen1, Zhen-Dong Li3,  
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

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


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.

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;14:1035-1040

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 2020 Feb 25 ];14:1035-1040
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Full Text


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}


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.


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).


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}{Figure 3}

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}

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 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}{Figure 7}

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.


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.


1Wang GS. Medical uses of mylabris in ancient China and recent studies. J Ethnopharmacol 1989;26:147-62.
2Yang H, Guo W, Xu B, Li M, Cui J. Anticancer activity and mechanisms of norcantharidin-Nd3II on hepatoma. Anticancer Drugs 2007;18:1133-7.
3Ho YP, To KK, Au-Yeung SC, Wang X, Lin G, Han X. Potential new antitumor agents from an innovative combination of demethylcantharidin, a modified traditional Chinese medicine, with a platinum moiety. J Med Chem 2001;44:2065-8.
4Peng C, Liu X, Liu E, Xu K, Niu W, Chen R, et al. Norcantharidin induces HT-29 colon cancer cell apoptosis through the alphavbeta6-extracellular signal-related kinase signaling pathway. Cancer Sci 2009;100:2302-8.
5Chen YJ, Tsai YM, Kuo CD, Ku KL, Shie HS, Liao HF. Norcantharidin is a small-molecule synthetic compound with anti-angiogenesis effect. Life Sci 2009;85:642-51.
6Yang PY, Chen MF, Kao YH, Hu DN, Chang FR, Wu YC. Norcantharidin induces apoptosis of breast cancer cells: Involvement of activities of mitogen activated protein kinases and signal transducers and activators of transcription. Toxicol In Vitro 2011;25:699-707.
7Chen YJ, Kuo CD, Tsai YM, Yu CC, Wang GS, Liao HF. Norcantharidin induces anoikis through Jun-N-terminal kinase activation in CT26 colorectal cancer cells. Anticancer Drugs 2008;19:55-64.
8Muggia FM, Braly PS, Brady MF, Sutton G, Niemann TH, Lentz SL, et al. Phase III randomized study of cisplatin versus paclitaxel versus cisplatin and paclitaxel in patients with suboptimal stage III or IV ovarian cancer: A gynecologic oncology group study. J Clin Oncol 2000;18:106-15.
9Reck M, von Pawel J, Zatloukal P, Ramlau R, Gorbounova V, Hirsh V, et al. Phase III trial of cisplatin plus gemcitabine with either placebo or bevacizumab as first-line therapy for nonsquamous non-small-cell lung cancer: AVAil. J Clin Oncol 2009;27:1227-34.
10Shen DW, Pouliot LM, Hall MD, Gottesman MM. Cisplatin resistance: A cellular self-defense mechanism resulting from multiple epigenetic and genetic changes. Pharmacol Rev 2012;64:706-21.
11Yu T, Hou F, Liu M, Zhou L, Li D, Liu J, et al. Norcantharidin anti-angiogenesis activity possibly through an endothelial cell pathway in human colorectal cancer. Asian Pac J Cancer Prev 2012;13:499-503.
12Kakar SS, Ratajczak MZ, Powell KS, Moghadamfalahi M, Miller DM, Batra SK, et al. Withaferin a alone and in combination with cisplatin suppresses growth and metastasis of ovarian cancer by targeting putative cancer stem cells. PLoS One 2014;9:e107596.
13Chen J, Wang J, Lin L, He L, Wu Y, Zhang L, et al. Inhibition of STAT3 signaling pathway by nitidine chloride suppressed the angiogenesis and growth of human gastric cancer. Mol Cancer Ther 2012;11:277-87.
14Cimmino F, Schulte JH, Zollo M, Koster J, Versteeg R, Iolascon A, et al. Galectin-1 is a major effector of TrkB-mediated neuroblastoma aggressiveness. Oncogene 2009;28:2015-23.
15Siddik ZH. Cisplatin: Mode of cytotoxic action and molecular basis of resistance. Oncogene 2003;22:7265-79.
16Galluzzi L, Senovilla L, Vitale I, Michels J, Martins I, Kepp O, et al. Molecular mechanisms of cisplatin resistance. Oncogene 2012;31:1869-83.
17Chen YN, Cheng CC, Chen JC, Tsauer W, Hsu SL. Norcantharidin-induced apoptosis is via the extracellular signal-regulated kinase and c-Jun-NH2-terminal kinase signaling pathways in human hepatoma HepG2 cells. Br J Pharmacol 2003;140:461-70.
18Luan J, Duan H, Liu Q, Yagasaki K, Zhang G. Inhibitory effects of norcantharidin against human lung cancer cell growth and migration. Cytotechnology 2010;62:349-55.
19Liao HF, Su SL, Chen YJ, Chou CH, Kuo CD. Norcantharidin preferentially induces apoptosis in human leukemic Jurkat cells without affecting viability of normal blood mononuclear cells. Food Chem Toxicol 2007;45:1678-87.
20Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57-70.
21Heath VL, Bicknell R. Anticancer strategies involving the vasculature. Nat Rev Clin Oncol 2009;6:395-404.
22Jain RK, Carmeliet P. SnapShot: Tumor angiogenesis. Cell 2012;149:1408.
23Carmeliet P. Angiogenesis in life, disease and medicine. Nature 2005;438:932-6.
24Kitadai Y. Angiogenesis and lymphangiogenesis of gastric cancer. J Oncol 2010;2010:468725.
25Svagzdys S, Lesauskaite V, Pavalkis D, Nedzelskiene I, Pranys D, Tamelis A. Microvessel density as new prognostic marker after radiotherapy in rectal cancer. BMC Cancer 2009;9:95.
26Zhang L, Ji Q, Liu X, Chen X, Chen Z, Qiu Y, et al. Norcantharidin inhibits tumor angiogenesis via blocking VEGFR2/MEK/ERK signaling pathways. Cancer Sci 2013;104:604-10.
27Zhang JT, Sun W, Zhang WZ, Ge CY, Liu ZY, Zhao ZM, et al. Norcantharidin inhibits tumor growth and vasculogenic mimicry of human gallbladder carcinomas by suppression of the PI3-K/MMPs/Ln-5γ2 signaling pathway. BMC Cancer 2014;14:193.
28Shen FZ, Wang J, Liang J, Mu K, Hou JY, Wang YT. Low-dose metronomic chemotherapy with cisplatin: Can it suppress angiogenesis in H22 hepatocarcinoma cells? Int J Exp Pathol 2010;91:10-6.
29Bjerkvig R, Johansson M, Miletic H, Camaj P, Eichhorn M, Ischenko I, et al. Cancer stem cells and angiogenesis. Semin Cancer Biol 2016;19:279-84.