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YangZheng XiaoJi capsule anti-tumour growth effects by antagonis

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Abstract

 

Background

Hepatocyte growth factor (HGF) is a cytokine that has a profound effect on cancer cells by stimulating migration and invasion and acting as an angiogenic factor. In lung cancer, the factor also plays a pivotal role and is linked to a poor outcome in patients. In particular, HGF is known to work in combination with EGF on lung cancer cells. In the present study, we investigated the effect of a traditional Chinese medicine reported in cancer therapies, namely YangZheng XiaoJi (YZXJ) on lung cancer and on HGF mediated migration and invasion of lung cancer cells.

 

Methods

Human lung cancer cells, SKMES1 and A549 were used in the study. An extract from the medicine was used. Cell migration was investigated using the EVOS and by ECIS. Cell–matrix adhesion and in vitro invasion were assessed. In vivo growth of lung cancer was tested using an in vivo xenograft tumour model and activation of the HGF receptor in lung tumours by an immunofluorescence method.

 

Results

Both lung cancer cells increased their migration in response to HGF and responded to YZXJ by reducing their speed of migration. YZXJ markedly reduced the migration and in vitro invasiveness induced by HGF. It worked synergistically with PHA665752 and SU11274, HGF receptor inhibitors on the lung cancer cells both on HGF receptor activation and on cell functions. A combination of HGF and EGF resulted in a greater increase in cell migration, which was similarly inhibited by YZXJ, and in combination with the HGF receptor and EGF receptor inhibitors. In vivo, YZXJ reduced the rate of tumour growth and potentiated the effects of PHA665752 on tumour growth. It was further revealed that YZXJ significantly reduced the degree of phosphorylation of the HGF receptor in lung tumours.

 

Conclusion

YZXJ has a significant role in reducing the migration, invasion and in vivo tumour growth of lung cancer and acts to inhibit the migratory and invasive effects induced by HGF and indeed by HGF/EGF. This effect is likely attributed to the inhibition of the HGF receptor activation. These results indicate that YZXJ has a therapeutic role in lung cancer and that combined strategy with methods to block HGF and EGF should be considered.

 

Background

 

Lung cancer is the leading cancer worldwide and results in more death than any other cancer types. In the UK, the incidence of lung cancer in males has steadily declined, but in contrast, the rate in females has steadily increased. Worldwide, although the incidence rate has been declining in developed countries, since the later part of the last century, the incidence continues to rise in countries in which smoking has peaked including China, Korea, and several countries in Africa and will continue to do so for the next few decades. Treatment options for lung cancer remain limited, which is reflected in the poor survival, namely 5 year survival remains below 10 % and 10 year survival close to 5 %. Surgery, chemotherapy and radiotherapy remain the main treatment choice, although new targeted therapies are now available, namely anti-EGFR, etc.

 

Recently, there have been early clinical reports that a Chinese medicinal formula, known as YangZheng XiaoJi (YZXJ), has a beneficial effect in patients with lung cancer. In a number of small trials, YangZheng XiaoJi, in combination with chemotherapy has been shown to increase the survival rate and at the same time, reduced the side effects. A similar beneficial effect has been reported in patients with primary hepatocellular carcinoma. Although it was initially proposed that the beneficial effects may be due to the improved immune function, such as the increase in NK cell functions, there have been recent reports to show that YangZheng XiaoJi was able to directly inhibit angiogenesis and migration of cancer cells, including osteosarcoma cells, an effect attributable to the inhibition on the activation of focal adhesion kinase.

 

Hepatocyte growth factor (HGF) is a cytokine that has strong effects on normal cells and cancer cells. In normal physiology, the cytokine is involved in tissue regeneration and organ repair, for example liver and lung regeneration. In cancer, however, the cytokine has been shown to have a profound effect on the migration, invasion and growth of cancer cells and has acted as a powerful angiogenic and lymphangiogenic factor [14, 15]. In the majority of solid tumour types, HGF and its receptor, cMET, have been found to be over-expressed in cancer cells and tumour tissues. It has been shown to be linked to disease progression, metastasis and long term clinical outcome of the patients.

 

In non-small cell lung cancer (NSCLC), HGF receptor protein over-expression has been frequently demonstrated and is shown to be associated with a poor clinical outcome of the patients. It has been shown that cMET protein expression is increased in NSCLC lung tumours with ALK gene rearrangement, and that gene amplification is uncommon in lung cancer. The amplified cMET protein expression may be the result of transcription factor ETS2 which was frequently down regulated in lung cancer.

 

In lung cancer, HGF has also been shown to interfere with EGF tyrosine kinase activation, which in turn results in induced resistance to EGFR inhibitor therapies. Thus, combined use of MET tyrosine kinase inhibitor (TKI) and EGF TKI has been suggested to be a valid novel combination to overcome TGF TKI acquired resistance in lung cancer. This was indeed shown in an in vitro study in which the cMET small inhibitor E7050 has the ability to circumvent resistance to the reversible, irreversible, and mutant-selective EGFR-TKIs induced by exogenous and/or endogenous HGF in EGFR mutant lung cancer cell lines, by blocking the Met/Gab1/PI3 K/Akt pathway in vitro. It is interesting to note that HGF-positive serum is a predictive factor for patients negative response to gefitinib therapy with advanced NSCLC who harbour wild-type EGFR. Serum HGF levels have been shown to be linked to disease progression and overall survival, and interestingly even more so when EGFR status was considered.

 

Previous reports have demonstrated a profound effect of YangZheng XiaoJi on the migration of cancer cells and indeed on endothelial cells, suggesting that the medicine is an important regulator for cell motility and potential target for therapy. HGF is one of the most powerful motogens (motility stimulating cytokines) which, together with the HGF receptor, cMET, are aberrantly expressed in lung cancer. In the present study, we sought to investigate the effects of YangZheng XiaoJi on the migration, invasion and cell adhesion of lung cancer cells, in particular in the context of stimulation by HGF. In this context, the degree of the activation/phosphorylation of the HGF receptor was also investigated in order to decipher if and how YangZheng XiaoJi might affect the HGF/cMET complex. The study also examined the effect of YangZheng XiaoJi on the growth of lung tumours in vivo. Here, we report that YangZheng XiaoJi has an inhibitory effect on the functions and growth of lung cancer cells, in vitro and in vivo. YangZheng XiaoJi also exhibited an effect on HGF- and HGF/EGF-induced cellular migration on lung cancer cells and impacted on the phosphorylation of the HGF receptor, cMET.

 

Methods

 

Cells and materials

Human lung cancer cell lines, SKMES1 and A549 were obtained from ATCC/LGC standard (Teddington, Middlesex, England, UK). Recombinant human hepatocyte growth factor was a kind gift from Professor Toshikazu Nakamura (Osaka University, Osaka, Japan). Recombinant human EGF was from Sigma Aldrich (Poole, Dorset, England, UK). Small inhibitors to HGF receptor, SU11274 (IC50 for cMET 20 nM) and PHA665752 (IC50 for cMET 9 nM), and small inhibitor to the EGFR kinase, AG490 (IC50 for EGFR 2 µM) were obtained from Tocris Chemicals (Bristol, England, UK). Antibodies to human HGF, human cMET (SC-10), and phospho-cMET (sc-34088) were obtained from Santa Cruz Biotechnologies Inc., (Santa Cruz, CA, USA). Horse radish peroxidase (HRP) conjugated fluorescently tagged secondary antibodies were obtained from SigmaAldrich (Poole, Dorset, England, UK).

 

The herbal medicinal formula, YangZheng XiaoJi was obtained from Yiling Pharmaceuticals (Shijiazhuang, HeBei, China). The formula contained the following 16 ingredients: Panax ginseng C.A. Mey., Astragalus memebranaceus (Fisch.) Bge.var. mongholicus (Bge.) Hsiao, Ligustrum lucidum Ait., Curcuma phaeocaulis Val., Ganodema lucidum, Gynostemma pentaphylla (Thunb) Mak, Atractylodes macrocephala Koidz, Scutellaria barbata D.Don, Oldenlandia diffusa (willd.) Roxb., Poria cocos, Duchesnea indica Focke, Solanum lyratum Thunb., Artemisia scoparia (Bge.) Ki, Cynanchum paniculatum Kitag, Eupolyphaga sinensis Walker, and Gallus domesticus Brisson. The chemical fingerprints of different batches were found to be highly consistent. Shown in Additional file 1 are fingerprints from 10 batches of YangZheng XiaoJi over a 2 year period (Additional file 1).

 

YangZheng XiaoJi extract, referred to as DME25, was prepared from YangZheng XiaoJi as previously reported using a DMSO based method. Briefly, YangZheng XiaoJi formulated powder was added to pure DMSO solution (Sigma Aldrich). This was placed on a rotating wheel (Labinco BV, Wolf Laboratory, York, England, UK) for 12 h in a cold room at 4 °C at 100 rpm [8, 10]. The preparation was centrifuged at 15,000×g, for 20 min at 4 °C. The supernatant was carefully removed and filtered using a disc filtration unit (pore size 0.20 μm, Sartorius Stedim, Sartorius, Epson, Surrey, England, UK). The extract was diluted in a balanced salt solution (BSS) and standardised by quantifying the optical density of the preparation using a spectrophotometer at 450 nm wavelength (Biotek, Wolf Laboratory). A master preparation which gave 0.25 OD at 450 nm was stored as the master stock and so named as DME25 for the subsequent experiments. DME25 at dilutions below 1:40 did not show an effect on the growth of lung cancer cells. In the present study, we used the dilution range between 1:100 to 1:2000.

 

 






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