| China has more than 4 million new cases of cancer every year, there are 2.8 million deaths per year, and the morbidity and mortality rates are still rising year by year. The family, state and society had got enormous burden from the grim situation. The highest morbidity and mortality rates of cancer is lung cancer, because of its high rates of metastasis, resulting in shorter survival of patients with poor prognosis after surgery should be dealt with seriously.With the widespread use of chemical anti-cancer drugs, drug resistance becomes a problem had to face it. Celastrol as a natural compound is extracted from herbal medicine, because of its high effectivity, low toxicity, and broad anti-cancer bioactivity has been attracted more and more attention. But it is also a lack of sufficient knowledge on suppress non-small cell lung cancer metastasis.According to reports, CD146 is involved in a large variety of tumor cells metastasis, invasion and angiogenesis. And CD146 e xpression level is closely related with on-small cell lung cancers’ disease progression and patient survival, and the level of soluble CD146 in non-small cell lung cancer patients’ plasma is also higher than normal, but at the cel ular level is still a lack of sufficient evidence.So we have A549 cells as experimental material and celastrol as experimental drugs, and try to study the role of celastrol in inhibiting A549 cell invasion and metastasis. 1. The main works are as following:(1) Cell activity of A549 cells were tested by MTT method afer induction of celastrol in 12 h, 24 h and 48 h.(2) Cell morphology was observed with AO/PI staining.(3) The migration of A549 cells was detected by wound healing assay.(4) The adhesion ability of A549 cells was detected by cell-adhesion assays.(5) The invasion ability of A549 cells was detected by Transwell.(6) The mRNA expression of CD146 and ERM family was detected by RT-PCR.(7) The proteins expression of CD146 was analyzed by Western Blot. 2. The results were as follows:(1) After induction of celastrol in A549 cells on 12 h, 24 h and 48 h, the cells activity were changed. With the increase of celastrol concentration, the A549 cells activity decreased, and showed a dose-and time-dependent manner. The IC50 of 24 h is 3.546±0.343μmol/L, and the IC50 of 48 h is 2.523±0.135μmol/L. Therefore, with 0.5μmol/L as the initial concentration to study the effect of celastrol on A549 cells metastasis.(2) Afer stained by AO/PI, we know that celastrol significantly effect the morphology of A549. With the increase of celastrol concentration, the cell membrane of A549 rupture, apoptotic bodies were observed, at last cel s dropped.(3) Through the cell scratch test, we examined the impact of celastrol on A549 cells migration. We know that celastrol can significantly inhibit A549 cells migration. With the increase of celastrol concentration, the relative migration distance decreases. When the concentration was 1.5μmol/L, the relative migration distance of A549 cells only took 17.88% of the control group.(4) Through the cell-adhesion assays, we know that with the increase of celastrol concentration, the adhesion rate of A549 cells decreased. When the concentration was 0.5μmol/L, the cell adhesion rate was 76.9%. When the drug concentration was increased to 2.0μmol/L, the cell adhesion rate was just 50.8%.(5) Through Transwell assays, we know that with the increase of celastrol concentration, the number of cells into the lower chamber gradually reduced. When the concentration was 1.5μmol/L, the number of cells into the lower chamber was only took 46.0% of the control group. Celastrol can significantly reduce the invasion ability of A549 cells.(6) Through RT-PCR assays, we know that celastrol can significantly reduce the expression of C D146 mRNA. With the drug effect, CD146-ERM complexes, we suspect that C146 through Moesin phosphorylation to affect downstream signaling, and affect the cel s metastasis.(7) Through Western Blot we know that celastrol can significantly reduce the expression of CD146 protein. When the concentration was 1.0μmol/L, the expression level of CD146 protein took 66.6% of the control group. And when the concentration was 1.5μmol/L, the expression level of CD146 protein took 36.7% of the control group.Through the above experimental results, celastrol significantly inhibit migration and invasion potency of A549 cells. The expression levels of CD146 mRN A and protein also decreased with the increase of celastrol concentration. In study, we provided some specific experimental evidence for further study ceslastrol inhibit the tumors metastasis. |
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