PEITC-containing pharmaceutical composition and application thereof in cancer therapy
A composition and drug technology, which can be used in drug combinations, medical preparations containing active ingredients, antitumor drugs, etc., and can solve problems such as poor prognosis in cancer treatment
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[0178] Preparation of lysate and Western blot analysis
[0179] Different lysates were used to prepare soluble, insoluble, and whole cell lysates. Cells were harvested by washing twice with 1x PBS and used to prepare lysates (soluble). RIPA buffer (10 mM sodium phosphate (pH 7.2), 300 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 1% deoxycholate, 2 mM EDTA) was added to the cells and placed in an ice bath for 30 minutes. The cell suspension was then centrifuged at 18500 xg for 10 min at 4°C and the supernatant was collected unless otherwise mentioned. The precipitate is the insoluble component. The insoluble fraction was dissolved in lysis buffer (65 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2% SDS, 50 mM DTT) containing 2% SDS. Harvested cell pellets were dissolved in 2% SDS lysis buffer to prepare whole cell lysis fractions as described above. The fractions were collected by centrifugation at 18500×g for 10 minutes at 4°C. Then take 30-50μg of the lysate for 4–12% SDS / PAGE electrophores...
Embodiment 1
[0203] The inhibitory effect of PEITC on the proliferation of mutant p53 cells
[0204] 1. PEITC inhibits the proliferation of prostate cancer cells by acting on DU145 mutant p53
[0205] The effect of PEITC on the proliferation of prostate cancer cell line DU145 was studied by WST-1 assay. For this, DU145 cells were treated with different concentrations of PEITC for 72 hours. After that, the corresponding IC50 was calculated by detecting the OD450 value of each group of cells. The results showed that PEITC reduced the proliferation of DU145 cell line (IC 50 s=~8μM)( figure 1 A).
[0206] 2. PEITC acts on breast cancer cells SK-BR-3, AU565 mutant p53 R175 Inhibit its proliferation
[0207] The WST-1 experimental study showed that PEITC can reduce the proliferation of various cells expressing mutant p53. Among them, PEITC expressed p53 R175 Mutant human breast cancer cells SK-BR-3, AU565 and human non-small cell lung cancer HOP92 have the strongest inhibitory effect. I...
Embodiment 2
[0212] PEITC inhibits tumor cell proliferation and induces apoptosis in a mutant p53-dependent manner
[0213] 1. PEITC to p53 R175 or p53 R248 Inhibition of proliferation and induction of apoptosis in breast cancer cell line SK-BR-3 in a dependent manner
[0214] In order to determine whether the effect of PEITC on inhibiting tumor cell proliferation is through making p53 R175 Transformation mediated by altered wild-type p53 activity, WST-1 assay after transfection of p53siRNA shows: p53 R175 SK-BR-3 cells with reduced expression ( figure 2 b) Significantly reduced sensitivity of PEITC to inhibit proliferation, while cells transfected with NS siRNA maintained high sensitivity. However, no significant difference in proliferation was observed in A549 cells in the same experiment ( figure 2 b). The above results indicated that PEITC-induced proliferation inhibition was dependent on mutant p53 protein.
[0215] In order to determine whether PEITC induces tumor cell apopt...
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