|Year : 2017 | Volume
| Issue : 4 | Page : 725-729
Effects of Macrothele raven venom on intrarenal invasion and metastasis of H22 liver cancer cells in mice
Yi Hou1, Xiaokun Zhao1, Jiaqin Chen2, Jingsheng Zhou2, Weiwei Chen2, Haifeng Mao3, Rui Chen2
1 Department of Urological Surgery, Second Xiangya Hospital, Central South University, Changsha 410012, PR China
2 Physical Education College, Hunan Normal University, Changsha 410012, PR China
3 Department of Physical Education, Yichun College, Yichun, Jiangxi 336000, PR China
|Date of Web Publication||13-Sep-2017|
Department of Urological Surgery, Second Xiangya Hospital, Central South University, Changsha 410012
Source of Support: None, Conflict of Interest: None
Background: Extrahepatic metastatic hepatocellular carcinoma (HCC) and its insensitivity to chemotherapy are the main causes of poor prognosis in patients with HCC. This study investigated the anti-cancer effect of Macrothele raveni venom on intrarenal metastatic HCC.
Materials and Methods: Subrenal capsule xenograft model of HCC was established by inoculation of H22 liver cancer cells.
Results: The general health, histology, and molecular changes were observed after administering 10 times of different dose of Macrothele raven venom injections. A volume of 0.8 μg/ml and 1.0 μg/ml of Macrothele raven venom significantly improved general health status in mice with subrenal capsule HCC tumors. Hematoxylin and eosin staining showed that Macrothele raven venom dose-dependently reduced invasion and metastasis of liver cancer cells in the kidney. Immunohistochemistry and real-time polymerase chain reaction showed that Macrothele raven venom injection dose-dependently decreased PI3K mRNA and protein, Akt protein, and mTOR mRNA expression, but increased Bad mRNA and protein expression in the kidney with H22 tumor cell invasion. 0.8 μg/ml is the most effective dose for the treatment of intrarenal metastatic HCC.
Conclusions: Macrothele raven venom dose-dependently inhibits invasion and metastasis of intrarenal metastatic HCC through inhibition of PI3K-Akt-mTOR signaling and increase of Bad expression.
Keywords: Akt, Bad, extrahepatic metastasis, hepatocellular carcinoma, invasion, Macrothele raven venom, mTOR, PI3K
|How to cite this article:|
Hou Y, Zhao X, Chen J, Zhou J, Chen W, Mao H, Chen R. Effects of Macrothele raven venom on intrarenal invasion and metastasis of H22 liver cancer cells in mice. J Can Res Ther 2017;13:725-9
|How to cite this URL:|
Hou Y, Zhao X, Chen J, Zhou J, Chen W, Mao H, Chen R. Effects of Macrothele raven venom on intrarenal invasion and metastasis of H22 liver cancer cells in mice. J Can Res Ther [serial online] 2017 [cited 2018 Aug 16];13:725-9. Available from: http://www.cancerjournal.net/text.asp?2017/13/4/725/214460
| > Introduction|| |
Hepatocellular carcinoma (HCC) is the fifth most common cancer and the second leading cause of cancer-related deaths worldwide. The prognosis of HCC is rather poor, and the reported 5-year survival in the US is only 12%. The poor prognosis of HCC can be attributed to most patients being diagnosed at an advanced intrahepatic tumor stage with extrahepatic metastasis. The most common sites of extrahepatic metastasis of HCC are the lung, abdominal lymph nodes, and bone. Renal metastasis from primary HCC is relatively rare. However, the molecular mechanisms driving the intrarenal metastasis remain to be fully elucidated. Moreover, there is currently no effective chemotherapy for metastatic HCC.
The composition of Macrothele raveni venom is very complex and consists of neurotoxic peptides, proteins, and low molecular weight compounds.In vitro studies have shown that Macrothele raveni venom can inhibit cell proliferation of human hepatoma BEL-7402 cells, HepG2 cells, and cervical cancer Hela cells. For example, treatment with Macrothele venom has been shown to strongly inhibit proliferation, increase apoptosis, arrest cells at G0/G1 phase, and reduce the length of S phase in BEL-7402 cells., A recent study also showed that spider toxin can inhibit subcutaneous H22 tumor growth. In addition, Macrothele raven venom was shown to be cytotoxic in HeLa cells. Moreover, spider venom can induce cell apoptosis and necrosis in MCF-7 cells. Furthermore, the venom of the spider Macrothele raven can induce apoptosis in myelogenous leukemia K562 cells. However, the molecular mechanisms involved in the anti-cancer effect of Macrothele venom remain to be elucidated, particularly in HCC.
PI3K/Akt/mTOR signaling pathway is a critical pathway that is widely studied in cancers. A variety of oncogenic effects of PI3K/Akt/mTOR signaling have been reported. For example, PI3K/Akt/mTOR signaling can promote tumorigenesis and cancer development, tumor angiogenesis, and cell secretion of vascular endothelial growth factor, which promotes the metastasis of a variety of malignant tumors. In addition, PI3K/Akt/mTOR signaling can accelerate the transition between theG1 and S phases of the cell cycle to promote cell proliferation. Proteins in the Bcl-2 family have now been divided into two categories according to their functions: One is anti-apoptosis genes, represented by Bcl-2; and the other is pro-apoptosis genes represented by Bad. Bad can form a Bad-Bcl-2 heterodimer with Bcl-2 protein to exert an antagonistic effect on Bcl-2. We hypothesize that Macrothele raven venom may regulate PI3K/Akt/mTOR signaling and Bad protein expression in extrahepatic metastatic HCC.
This study investigated the effect of Macrothele raven venom on intrarenal metastasis in a model of H22 hepatoma cell invasion on kidney membrane and explored associated molecular mechanisms.
| > Materials and Methods|| |
Subrenal capsule xenograft model
Sixty healthy male Kunming mice, weighing 18–22 g, were provided by the X Laisikegenda company (X place). Mice were anesthetized by intraperitoneal injection of 10% chloral hydrate. The left dorsal skin was disinfected. Skin and muscles were cut using scissors. An oblique incision (<1 cm) was made, and the left kidney was uncovered by tweezers. 0.05 ml of H22 cell suspension (1 × 103/ml) was seeded under the renal capsule using a sterile syringe. The kidney was placed back into the mouse, and the incision was sutured. Mice with successful injection of H22 cells were randomly divided into 6 groups: No Macrothele raven venom, 0.2 μg/ml of venom, 0.4 μg/ml of venom, 0.6 μg/ml of venom, 0.8 μg/ml of venom, and 1 μg/ml of Macrothele raven venom (n = 10). All mice were housed at the animal facility of Hunan Normal University and were given free access to food and water.
The animal study protocol was approved by the Institutional Animal Care Committee of Hunan Normal University and was carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Macrothele raven venom injection
Macrothele raven venom solution was provided by the Department of Protein Chemistry and Molecular Biology, the Life Science College of Hunan Normal University and diluted with saline (1:10) before use. Injection was initiated on the third day after surgery through tail vein. Macrothele raven venom was given at 0, 0.2, 0.4, 0.6, 0.8, and 1.0 μg/ml every other day for 20 days (10 injections).
After 10 injections, the mice were subjected to fasting overnight, anesthetized with chloral hydrate, and killed by cervical dislocation. Tumors were excised, weighed, and cut into two pieces. One piece of the tumor tissue was snap frozen in liquid nitrogen for total RNA extraction, and the other piece of tissue was placed in 4% paraformaldehyde solution, fixed for more than 12 h, dehydrated, and then paraffin embedded.
Hematoxylin and eosin staining
The paraffin tissues were sectioned at 4 μm thickness and deparaffinized. After rinsed with 95% alcohol for three times × 2 min, rinsed under running water for 10 min, and dipped in 95% alcohol once, sections were treated with hematoxylin for 20 min, 1% hydrochloric acid for 2 s, and distilled water for 3 min. After treatment with saturated lithium carbonate for 10 s and running water for 10 min, sections were stained with eosin for 10s. After washing with running water for 10 min, sections were rinsed with 95% alcohol for three times × 2 min, treated with xylene, and mounted on slides. Histological and morphological changes were observed under Olympus (Jarm, Japan) Optical microscopy.
Immunohistochemical staining was performed using SABC kits (Wuhan Boster Biotechnology Company, Wuhan, China). The primary PI3K, AKT, and Bad antibodies were purchased from Wuhan Boster Biotechnology Company. Briefly, 4 μM thick paraffin-embedded sections were deparaffinized and treated with routine antigen retrieval. The sections were then incubated with primary antibody (1:300 dilution) at room temperature for 1 h, washed with 1X phosphate buffered saline (PBS) for three times, and then incubated with secondary antibody (1:1000) for 30 min. After washing with 1X PBS and staining with 3,3-diaminobenzidine tetrahydrocloride, slides were prepared as described above. PBS was used in replace of primary antibody as a negative control. Simple PCI image analysis system (C-imaging Company, USA) was used to calculate the percentage of positive staining area in each slide.
Real-time polymerase chain reaction
Total RNA was extracted from 20 mg of tumor tissues using Animal Tissue RNA Extraction kit (Tiangen Company, Beijing, China). RNA was dissolved in DEPC treated water, and the concentration was adjusted to 1 μg/μl. Reverse transcription was performed using Reverse Transcription kit (Invitrogen, USA). Real-time polymerase chain reaction (PCR) was performed using ABI 7900HT Real-time PCR instrument. The following real-time PCR reaction parameters were used: 95°C, 30 s followed by 40 cycles of 95°C, 5 s and 60°C, 30 s. Primers of PI3K, mTOR, and Bad genes were purchased from Shanghai Sangon Company (Shanghai, China).
Data were presented as the mean ± standard deviation and analyzed using SPSS19.0 software (IBM, USA). Differences between groups were analyzed using one-way ANOVA and two-tails t-test pairwise comparisons. P < 0.05 was considered statistically significant.
| > Results|| |
General observation in animals
All mice survived until the end of the observation. Mice that were treated with 0.8 and 1.0 μg/ml of Macrothele raven venom exhibited normal activity and no weight loss compared to normal control mice. Mice that were treated with 0, 0.2, 0.4, and 0.6 μg/ml of Macrothele raven venom showed obvious ascites and peritoneal tumor metastasis. In contrast, mice that received 0.8 and 1.0 μg/ml of Macrothele raven venom exhibited a small amount of ascites and light peritoneal metastases.
Kidney tissue morphology
[Figure 1] shows that the kidney tissue morphology in mice that received no Macrothele raven venom treatment (0 μg/ml) was irregularly arranged with visible metastasis of tumor cells, unclear boundaries between kidney tissue and tumors, and serious invasion and metastasis. In the kidney of mice that received 0.2– 0.6 μg/ml of Macrothele raven venom, the tumor cell metastasis and invasion decreased in a dose-dependent manner. No obvious metastasis or invasion of tumor cells in the kidney was observed in mice that received 0.8 μg/ml and 1.0 μg/ml of Macrothele raven venom.
|Figure 1: Hematoxylin and eosin staining of kidney tissues in mice (×200). (a) Mice received no Macrothele raven venom treatment (0 μg/ml). (b-f) Mice received 0.2 μg/ml (b), 0.4 μg/ml (c), 0.6 μg/ml (d), 0.8 μg/ml (e), and 1.0 μg/ml (f) of Macrothele raven venom treatment, respectively. The concentration of 0.8 μg/ml was the highest efficacy. ↓ indicates invasion and indicates metastasis|
Click here to view
PI3K, AKT, and Bad protein and PI3K, mTOR, and Bad mRNA expression in renal tissues
Immunohistochemical staining showed that positive PI3K, AKT, and Bad staining was mainly located at the cytoplasm [Figure 2]. Bad expression was dose-dependently increased, whereas the PI3K and Akt protein expression was dose-dependently decreased after treatment with 0.0–1.0 μg/ml of Macrothele raven venom [Figure 2].
|Figure 2: Immunohistochemical staining of kidney tissue inoculated with H22 tumor cells (×200). (a) Mice received no Macrothele raven venom treatment (0 μg/ml). (b-f) Mice received 0.2 μg/ml (b), 0.4 μg/ml (c), 0.6 μg/ml (d), 0.8 μg/ml (e), and 1.0 μg/ml (f) of Macrothele raven venom treatment, respectively. (g1-g3) Negative control. a1–f1: PI3K expression; a2–f2: Akt expression; a3–f3: Bad expression. ↓ indicates positive staining|
Click here to view
Semi-quantitative analysis of immunohistochemical staining showed no significant differences in PI3K, Akt, and Bad expression in the kidneys of mice that received injection of 0.8 and 1.0 μg/ml of Macrothele raven venom compared to healthy controls (P > 0.05). PI3K expression was significantly lower in the kidneys of mice that received an injection of 0.8 μg/ml of Macrothele raven venom compared to other low dose groups (P < 0.01 or P < 0.05). Akt expression was significantly lower in the kidneys of mice that received injection of 0.8 μg/ml group compared to mice that received an injection of 0.4, 0.2, and 0 μg/ml of Macrothele raven venom (P < 0.01 or P < 0.05). Bad expression was significantly higher in the kidneys of mice that received 1.0 μg/ml of Macrothele raven venom compared to all other low dose groups (P < 0.01) [Table 1].
|Table 1: Percentage of positive PI3K, Akt, and Bad expression in immunohistochemistry of H22 tumor tissues|
Click here to view
Real-time PCR showed significantly higher PI3K and mTOR mRNA expression in the kidneys of mice that received an injection of 0.8 μg/ml of Macrothele raven venom compared to all other low dose groups (P < 0.01 or P < 0.05). Macrothele raven venom inhibited PI3K and mTOR mRNA expression in a dose-dependent manner. In contrast, Bad mRNA expression was significantly lower in the kidneys of mice that received 0.8 μg/ml of Macrothele raven venom compared to all other low dose groups (P < 0.01 or P < 0.05). Macrothele raven venom significantly increased Bad mRNA expression in a dose-dependent manner [Table 2].
|Table 2: PI3K, mTOR, and Bad mRNA expression (RQ value) in H22 tumor tissues|
Click here to view
The dose-response curve of Macrothele raven venom
The treatment efficacy was defined as the percentage of inhibition in tumor metastasis and invasion according to the hematoxylin and eosin (HE) staining, and pathological examination referring to the control group received 0 μg/ml Macrothele raven venom (no treatment). A dose-response curve was plotted [Figure 3]. The best dose with the highest efficacy was 0.8 μg/ml.
|Figure 3: The dose-response curve. The best dose for efficacy was 0.8 μg/ml. E: Efficacy; C: Concentration|
Click here to view
| > Discussion|| |
The toxicity of spider venom has been studied since the ancient times, and most spider venoms are highly stable and resistant to proteolytic degradation. Moreover, spider venoms from a variety of spiders have high specificity and potency for target therapy. The combination of bioactivity and stability has made spider-venom valuable as chemotherapy agents. However, the anti-cancer effect of Macrothele raven venom on extrahepatic metastatic HCC has not been reported. In this study, a subrenal capsule xenograft model of liver cancer was successfully established using H22 liver cancer cells. The subrenal capsule xenograft model was used to evaluate the anti-cancer activity of Macrothele raven venom on extrahepatic metastatic HCC. Results showed that Macrothele raven venom significantly inhibited invasion and metastasis of H22 liver cancer cells in the kidneys of subrenal capsule xenograft mice in a dose-dependent manner. The anti-cancer activity of Macrothele raven venom significantly correlated with the inhibition of PI3K-Akt-mTOR signaling and the increase in Bad expression.
In this study, the cage side observation showed significant improvement in general health status, including basic activities, hair color, diarrhea, and weight, of mice with subrenal capsule HCC tumors that injected with 0.8 and 1.0 μg/ml of Macrothele raven venom. HE staining of kidneys inoculated with H22 liver cancer cells demonstrated that Macrothele raven venom dose-dependently reduced invasion and metastasis of liver cancer cells in the kidney. 0.8 μg/ml of Macrothele raven venom almost inhibited intrarenal invasion and metastasis. Immunohistochemistry results showed that Macrothele raven venom injection dose-dependently decreased PI3K and Akt protein expression, but increased Bad protein levels in the mice kidneys with H22 tumor cell invasion. Real-time PCR showed that Macrothele raven venom injection dose-dependently decreased PI3K and mTOR mRNA expression, but increased Bad mRNA expression. Observations from the general health status, histology, and molecular changes suggest no significant differences in efficacy between 0.8 and 1.0 μg/ml of Macrothele raven venom. 0.8 μg/ml dose of Macrothele raven venom showed the highest efficacy for the treatment of intrakidney metastatic HCC.
A previous study demonstrated that Macrothele raven venom has a strong inhibitory effect on human hepatoma cells through interfering with cellular DNA synthesis to arrest cells at the G0/G1 phase and reduce the length of S phase. PI3K/Akt/mTOR signaling pathway plays a crucial role in cancer biology. mTOR is a major regulatory switch of cell metabolism, and mTOR inhibition may lead to stagnation of the early G1 cell cycle.,, We proposed that Macrothele raven venom may exert an anti-cancer effect in extrahepatic metastatic HCC through inhibition of the PI3K/Akt/mTOR signaling pathway, leading to arrest of cell cycle at G1-S phase and inhibition of cell proliferation. At the same time, Macrothele raven venom upregulated Bad protein level to antagonize the anti-apoptotic effects of Bcl-2 and induce tumor cell apoptosis. Therefore, Macrothele raven venom might block tumor cell cycle and activate apoptotic factors to inhibit tumor growth.
| > Conclusions|| |
Macrothele raven venom can inhibit invasion and metastasis of tumor cells in the subrenal capsule xenograft model of liver cancer in a dose-dependent manner. Macrothele raven venom exerts anti-cancer effects through inhibition of PI3K-Akt-mTOR signaling and increasing Bad expression.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Faltermeier C, Busuttil RW, Zarrinpar A. A surgical perspective on targeted therapy of hepatocellular carcinoma. Diseases 2015;3:221-52.
Aron M, Nair M, Hemal AK. Renal metastasis from primary hepatocellular carcinoma. A case report and review of the literature. Urol Int 2004;73:89-91.
Katyal S, Oliver JH 3rd
, Peterson MS, Ferris JV, Carr BS, Baron RL. Extrahepatic metastases of hepatocellular carcinoma. Radiology 2000;216:698-703.
Escoubas P, Diochot S, Corzo G. Structure and pharmacology of spider venom neurotoxins. Biochimie 2000;82:893-907.
Gao L, Shen JB, Sun J, Shan BE. Effect of the venom of the spider Macrothele raveni on the expression of p21 gene in HepG2 cells. Sheng Li Xue Bao 2007;59:58-62.
Gao L, Wei F, Shan B. The inhibitory effects of Reye spider venom on human hepatocellular carcinoma BEL-7402 cell proliferation and its mechanism. Cancer 2005;24:812-5.
Sheng ZJ, Qin CJ, Wei CW, Miao LC, Hua ZG, Rui C, et al.
The effect of aerobic exercise and Macrothele raveni venom on tumor-bearing mice. Int J Sports Med 2015;36:93-100.
Gao L, Shan BE, Chen J, Liu JH, Song DX, Zhu BC. Effects of spider Macrothele raveni venom on cell proliferation and cytotoxicity in HeLa cells
. Acta Pharmacol Sin 2005;26:369-76.
Gao L, Yu S, Wu Y, Shan B. Effect of spider venom on cell apoptosis and necrosis rates in MCF-7 cells. DNA Cell Biol 2007;26:485-9.
Liu Z, Zhao Y, Li J, Xu S, Liu C, Zhu Y, et al.
The venom of the spider Macrothele raveni induces apoptosis in the myelogenous leukemia K562 cell line. Leuk Res 2012;36:1063-6.
Yang L, Dan HC, Sun M, Liu Q, Sun XM, Feldman RI, et al.
Akt/protein kinase B signaling inhibitor-2, a selective small molecule inhibitor of Akt signaling with antitumor activity in cancer cells overexpressing Akt. Cancer Res 2004;64:4394-9.
Brooks C, Dong Z. Regulation of mitochondrial morphological dynamics during apoptosis by Bcl-2 family proteins: A key in Bak? Cell Cycle 2007;6:3043-7.
Zinkel S, Gross A, Yang E. BCL2 family in DNA damage and cell cycle control. Cell Death Differ 2006;13:1351-9.
Pineda SS, Undheim EA, Rupasinghe DB, Ikonomopoulou MP, King GF. Spider venomics: Implications for drug discovery. Future Med Chem 2014;6:1699-714.
Saez NJ, Senff S, Jensen JE, Er SY, Herzig V, Rash LD, et al.
Spider-venom peptides as therapeutics. Toxins (Basel) 2010;2:2851-71.
Proud CG. Regulation of mammalian translation factors by nutrients. Eur J Biochem 2002;269:5338-49.
Peng T, Golub TR, Sabatini DM. The immunosuppressant rapamycin mimics a starvation-like signal distinct from amino acid and glucose deprivation. Mol Cell Biol 2002;22:5575-84.
Beugnet A, Tee AR, Taylor PM, Proud CG. Regulation of targets of mTOR (mammalian target of rapamycin) signalling by intracellular amino acid availability. Biochem J 2003;372(Pt 2):555-66.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]