【Abstract】 Objective To study the histone deacetylase inhibitor, sodium butyrate, and to explore the molecular mechanisms of its anti-tumor effect of prostate cancer LNCaP cells HER 2 signaling pathway. Method tetramethyl thiazolyl tetrazolium (MTT) to detect the effects of drugs on tumor cell proliferation; hoechst 33342 staining morphological changes of cell apoptosis, Western blot detection of apoptosis marker protein, HER2 / neu, Phos Akt Phos protein of Erk peer signal. Results of sodium butyrate able to effectively inhibit proliferation of LNCaP cells and induce apoptosis the half effective killing dose (EC50) of 5.6mmol / L; drugs that inhibit HER 2 gene transcription and protein expression, and inhibition of downstream signaling pathways MAPK and AKT activation. Conclusion Sodium butyrate can play an inhibitory effect on tumor cells by blocking the growth of prostate cancer cells HER 2 has an important role in the signaling pathway.
Key words】 sodium butyrate; histone deacetylase inhibitor; prostate cancer; HER 2; cellular signaling pathways; apoptosis
Targeting HER 2 Signal Pathway in LNCaP Prostate Cancer Cells by Sodium Butyrate
HAO Tong li, XIAO Xu ren, GUO Gang, ZHU Jie
Department of Urology, PLA General Hospital, Beijing 100853, ChinaAbstract: Objective To investigate the mechanisms underlying the antitumor effect of histone deacetylase inhibitor sodium butyrate on LNCaP prostate cancer cells.Methods Proliferation of LNCaP cells exposed to sodium butyrate was detected by MTT assay. Cell apoptosis was assayed by hoechst 33342 nuclei staining as well as apoptosis marker protein leaved PARP expression. The expression of HER 2 as well as downstream key signaling molecules was assayed by western blotting after sodium butyrate exposure.Results Sodium butyrate killed LNCaP cells with an EC50 value of 5.6mmol / L within 48 hours as well as inducing cell apoptosis. Sodium butyrate could downregulate HER 2 expression through suppression of HER 2 gene transcription.Activation of downstream signaling molecules such as Akt and Erk were also suppressed.Conclusion Sodium butyrate exhibits significant antitumor effect against LNCaP cells through suppression of HER 2 signaling pathway.
Key words: Sodium butyrate; Histone deacetylase inhibitor; Prostate cancer; HER 2; Cell signaling pathway; Apoptosis
Sodium butyrate dietary fiber after glycolysis in the gut produce mainly short chain fatty acids, with the histone deacetylase inhibitor activity, mainly by changing the degree of acetylation of histones to change chromatin structure, involved in a variety of gene expressed. Animal experiments and clinical trials show that sodium butyrate can inhibit cell growth, induction of cell maturation, induce cancer cell apoptosis to treat tumors .
Recent studies have observed the sodium butyrate inhibitory effect on prostate cancer cell lines, but its mechanism is not clear, this inhibition was not clear of the androgen receptor , the experimental study of sodium butyrate on HER 2 signaling pathway, and to explore the mechanism of inhibition of prostate cancer LNCaP cells.
1 Materials and methods
The RPMI 1640, fetal bovine serum were purchased from Invitrogen Corporation; to trypsin, green streptomycin, L glutamine, sodium pyruvate was purchased from Hyclone company; sodium butyrate was purchased from Sigma, dissolved in PBS and stored at -20 ° C ; MTT (methyl thiazolyl tetrazolium blue) hoechst 33342 was purchased from Sigma; by mouse anti-β-actin, anti-poly (ADP ribose) polymerase degradation products (cleaved PARP) monoclonal antibody, horseradish peroxide was enzyme-labeled rabbit anti-mouse IgG products are Cell Signaling; BCA protein quantitation kit, chemiluminescent substrate purchased from PIERCE Company. RT PCR kit was purchased from Takara. The primer sequences are as follows: HER 2: 5 ‘ the AGC CGC GAG CAC the CCA AGT 3’, 5 ‘ TTG GTG GGC AGG TAG GTG AGT T 3’ ; Oligo 6.0 software design β2-microglobulin (β2 mG) primer sequences: 5 ‘ CTC the GCG CTA CTC TCT CTC TTT CTG G 3’, 5 ‘ GCT the TAC ATG TCT CGA TCC CAC TTA A 3’. Primer synthesis by on Boya biotechnology company. The DL600 Takara products.
1.2 cell lines and cell culture
LNCaP cells were kept in our laboratory, the medium containing 10% fetal calf serum, 2 mM L glutamine, 100U/ml penicillin, 100μg/ml streptomycin, 1.0mm sodium pyruvate RPMI 1640, at 37 ℃ saturated humidity and 5% CO2 under the conditions of cultivation.
1.3 Cell proliferation assay
By MTT assay. 104 cells were seeded in 96-well plates added to each well 5mg/ml by MTT 20μl incubated for 5h after lysate, 37 ℃ overnight, after the shock to Immunoskan340 enzyme-linked instrument 540nm wavelength detection absorbance. In each of three wells. Cell viability (%) = absorbance of experimental group / absorbance of control group × 100%.
1.4 Detection of apoptosis
Cell smears fixed with methanol, washed in PBS, hoechst 33342 staining, fluorescence microscope (Nikon ECLIPSE TE2000 U) nuclear morphology observed under UV excitation light.
1.5 Western blot detection
Logarithmic phase cells treated with drugs 48h (control group Add PBS) and cells were collected after Laemmli Lysates under ice cells were lysed for whole cell lysate, after thermal denaturation to the BCA kit protein quantification, taking 60μg protein samples were separated by polyacrylamide gel electrophoresis and then electrically transferred to PVDF membranes with 5% skim milk at room temperature closed 1H, plus an anti overnight, rinsing plus skim milk diluted horseradish peroxidase labeled secondary antibody reaction at room temperature for 1h, chemiluminescent chromogenic substrate, film exposure.
1.6 RT PCR
, Total RNA was extracted by Trizol kit instructions for reverse transcription reaction, followed by 35 cycles of PCR amplification of Her 2/neu 0.1μg/μl ethidium bromide-containing reaction product of a 2% agarose The gel electrophoresis was performed, JS 380 gel image analysis scanned.
1.7 statistical methods
The probit calculated half effective killer dose (EC50).
2.1 sodium butyrate on cell killing effect
96-well plates in LNCaP cells were grown to logarithmic phase, diluted according to the times than the concentration of sodium butyrate, 48h after the MTT assay cell survival situation, the probit method EC50 for 5.6mmol / L, as shown in Figure 1.
2.2 sodium butyrate-induced apoptosis of LNCaP cells
Drug action after 48h the cells were collected, hoechst 33342 staining, as shown in Figure 2, sodium butyrate treatment group found that part of the cell nucleus fragmentation, chromatin condensation, in line with the morphological changes of apoptosis. LNCaP cells exposed to sodium butyrate was measured after 48h apoptosis marker proteins – PARP degradation of product (Cleaved PARP), shown in Figure 2c, the control group PARP 116kD, and drug treatment appeared after 48h 89kd a degradation product, eligible apoptotic biochemical changes.
2.3 sodium butyrate of HER 2 impact
Western blot analysis showed that the drug treatment HER 2 protein expression in a dose-dependent decrease of 20 mmol / L sodium butyrate 48h able to completely inhibit the expression of HER 2.
2.4 sodium butyrate HER 2 gene transcription
RT PCR, 10 mmol / L sodium butyrate 24h beginning HER 2 mRNA levels significantly decreased, indicating that the drug can inhibit HER 2 gene transcription, as shown in Figure 3.
2.5 sodium butyrate inhibition of the MAPK and AKT activation
Sodium butyrate treatment Phos of Erk Phos Akt expression in a dose-dependent decrease of total Akt did not change significantly after the drugs inhibit HER 2 expression downstream MAPK and PI3K signaling pathway activation of key proteins (Phos Erk, Phos Akt) also been suppressed, as shown in Figure 4.
This study shows that sodium butyrate can inhibit HER-2 expression in LNCaP cells, and block the downstream activation of MAPK and PI3K Akt pathway of cell killing effect.
of her 2/neu gene (also known as c erbB2) encoding transmembrane receptor protein tyrosine kinase, is a member of the epidermal growth factor receptor family, have high expression in a variety of tumors. Normal prostate epithelial cells with low expression of HER 2, in prostate cancer, especially in androgen-independent prostate cancer increased expression . HER 2/neu receptor protein tyrosine kinase, can occur in the case of the absence of extracellular ligand itself activate MAPK, PI3K/Akt pathway promotes cell proliferation and apoptosis resistance [5 , 6]. In addition, of HER kinase signaling cascade activation is still capable of activation of the androgen receptor in prostate cancer cells resistant to inhibition of androgen receptor plays an important role in the progression of androgen-independent prostate cancer 7].
Previous studies have observed to sodium butyrate has inhibitory effect on prostate cancer, but there is no change in the expression of the androgen receptor drug action, illustrate that the effect is not mediated by the androgen receptor signaling pathway . But for HER 2 signaling pathway in which the role of no studies have been reported. The first study found that sodium butyrate can effectively inhibit the expression of HER 2 expression, thereby inhibiting key protein activation of downstream signaling pathways, blocking MAPK and PI3K/Akt signaling pathways to induce apoptosis in prostate cancer. Sodium butyrate inhibits HER 2 expression one of the mechanisms that inhibition of HER 2 gene transcription to promote HER 2 protein degradation is not clear. HER 2 play an important role in androgen-independent prostate cancer progression, sodium butyrate on gene transcription and protein expression levels of this key signaling protein expression inhibition, theoretically for the treatment of androgenic non-independent prostate cancer provides a new way of thinking.
Inadequacies of sodium butyrate is relatively inefficient (role of concentration mmol), rapid plasma clearance, making the sodium butyrate clinical applications are subject to certain restrictions. People develop a low toxicity and a more specific derivatives wherein AN 9 has been shown in clinical trials of a clear anti-tumor effects the prospects . Can be predicted to be used alone or sodium butyrate and its derivatives, or with androgen deprivation therapy, and there will be more extensive research and application of value in the treatment of advanced prostate cancer.