Study On Arsenic Trioxide Re-sensitizes Estrogen Receptorα-negative Breast Cancer Cells To Endocrine Therapy

Breast cancer is one of the most common malignant tumors in women and with the increasing incidence. It takes serious harm to the women’s health. In the past20years, breast cancer incidence was with an increasing trend in china although it was low before. The incidence rate jumps ranking first in the female malignancy incidence in the Shanghai area.30%～40%of breast cancers lack ERa, these tumors are associated with poorer histological differentiation, the higher postoperative recurrence and metastasis, and rarely respond to endocrine therapy such as tamoxifen (TAM), there is no effective therapeutic other than chemotherapy. Recent studies indicate that gene deletions, mutations and other genetic alterations of ERa are low incidence, suggesting that loss of the ERa expression is associated with the abnormal hypermethylation of the promoter CpG island.Arsenic trioxide (As2O3) is more and more important in traditional Chinese medicine in malignant cancer therapy. In recent years, it was reported that it had demethylation function by competitively using S-adenosylmethionine and direct or indirect inhibition of methyltransferase enzymes. Therefore, arsenic trioxide may be used as a demethylating agent to re-sensitize Estrogen Receptor a-negative breast cancer patents to endocrine therapy by restoring ERa re-expression. The literatures related treatment of malignant cancer by arsenic trioxide is much, but it is not common in treatment of breast cancer, and we are the first time to explorer the Arsenic trioxide re-sensitizes Estrogen Receptor a-negative breast cancer cell to endocrine therapy in vitro and in vivo.Part1The correlation of DNMT1and ERa expression with ERa methylation and its clinical pathological significance in normal breast specimens and breast cancer specimensMaterials and methods1. Immunohistochemistry and RT-PCR were used to detect the protein and mRNA expression of DNA methyltransferase-1(DNMT1) and ERa in20normal breast specimens and112breast cancer specimens.2. MS-PCR was used to detect the methylation status of ERa in20normal breast specimens and ERa-positive and ERa-negative breast cancer specimens.3. Statistical treatment:Statistical analyses were performed using the SPSS17.0package. The results were expressed as mean±standard deviation (x±s). The χ2test and Spearman rank correlation coefficient analysis were used to assess the univariate association between the correlation of DNMT1expression with ERa methylation and its clinical pathological significance in normal breast specimens and breast cancer specimens. P value<0.05was considered statistically significant.Results1. The Immunohistochemistry and RT-PCR results showed that the positive rates of DNMT1protein and mRNA were low in normal breast specimens and ERa-positive breast cancer specimens, which were10.00%、46.05%and15.00%、48.68%respectively, while it was high in ERa-negative breast cancer specimens, which were81.11%and88.89%respectively (P<0.05).2. MS-PCR results showed that:the methylation rates of ERa was increased by turns in normal breast specimens, ERa-positive breast cancer specimens and ERa-negative breast cancer specimens, which were10.00%,36.84%and86.11%(P<0.05).3. The positive expression of ERa protein was associated with the PR (P<0.05), but not with the Age, Menopause state, Tumor size, Number of Positive nodes, TNM stage, Tumor type (P>0.05). The protein and mRNA expression of DNMT1was negatively correlated to the expression of ERa (P<0.05). The expression of DNMT1was positively correlated to the methylation of ERa (P<0.05).Part2The demethylation effect and the impact on prolifation of Arsenic trioxide on ERa-negative breast cancer cells MDA-MB-435s in vitroMaterials and methods1. MS-PCR and Methylation sequencing was used to detect the methylation status of ERa in ERa-negative breast cancer cell line MDA-MB-435s after treated with different concentrations of arsenic trioxide (As2O3). Methylation sequencing showed that part of unmethylation Cytosines changed to Thymines.2. RT-PCR was used to detect the mRNA expression of DNA methyltransferase-1(DNMT1) and ERa in ERa-negative breast cancer cell line MDA-MB-435s after treated with different concentrations of arsenic trioxide (As2O3).3. Immunohistochemistry and western blot were used to detect the protein expression of DNA methyltransferase-1(DNMT1) and ERa in ERa-negative breast cancer cell line MDA-MB-435s after treated with different concentrations of arsenic trioxide (As2O3).4. Cell proliferation was examined using the MTT assay in ERa-negative breast cancer cell line MDA-MB-435s after treated with different concentrations of arsenic trioxide (As2O3).5. Statistical treatment:Statistical analyses were performed using the SPSS17.0package. The results were expressed as mean±standard deviation (x±s).The relative expression of DNMT1and ERα analysis was made using one way ANOVA test. P value<0.05was considered statistically significant.Results1. We observed the unmethylation specific bands of ERα gene were enhanced after treated by As2O3in concentration-dependently. 2. RT-PCR, Immunohistochemistry and Western blot results showed that the marked increase in ERa re-expression was accompanied by a significant reduction in DNMT1in a concentration-dependent manner compared with the untreated control group (P<0.05)3. The MTT assay showed that the level of proliferation in the ERa-negative breast cancer cell MDA-MB-435s was significantly suppressed after treatment with different concentrations of As2O3for24h and48h (P<0.05). Addition of TAM for an additional24h led to further suppression of MDA-MB-435s’proliferation.Part3The effect of Arsenic trioxide alone or combined with TAM on the tumor formation of MDA-MB-435s cell in nude miceMaterials and methods1. The volumes and weights of tumors were compared with the As2O3treated and normal saline group in human breast cancer cell xenografts in nude mice.2. MS-PCR and Methylation sequencing was used to detect the methylation status of ERα in in the As2O3treated and normal saline group in human breast cancer cell xenografts in nude mice. Methylation sequencing showed that part of unmethylation Cytosines changed to Thymines.3. RT-PCR, Immunohistochemistry and Western blot were used to detect the mRNA and protein expression of DNA methyltransferase-1(DNMT1) and ERa in in the As2O3treated and normal saline group in human breast cancer cell xenografts in nude mice.4. Statistical treatment:Statistical analyses were performed using the SPSS17.0package. The results were expressed as mean±standard deviation (x±s). The relative expression of DNMT1and ERa analysis was made using one way ANOVA test. P value<0.05was considered statistically significant.Results1. An obviously decrease in the volumes and weights of tumors in the As2O3treated group compared with the normal saline group. A further reduction in tumor size was observed after combined treatment of As2O3with TAM compared to the normal saline and treated with TAM alone groups (P<0.05).2. We observed a decrease in the ERa methylation specific bands in Arsenic trioxide alone or combined with TAM groups. In contrast, the unmethylation specific bands were enhanced. In addition, we found that the hypermethylated status of normal saline groups remains unchanged.3. RT-PCR, Immunohistochemistry and Western blot results showed that the marked increase in ERa re-expession was accompanied by a significant reduction in DNMT1expression in a concentration-dependent manner. We also found that the DNMT1and ERa were not changed in normal saline group and treated with TAM alone group (P<0.05).Conclusion1. In breast cancer specimens, the protein and mRNA expression of DNMT1was negatively correlated to the expression of ERa; the expression of DNMT1was positively correlated to the methylation of CpG island of ERa.2. ERa could be re-expressed in ERa-negative breast cancer cell MDA-MB-435s after treated with As2O3by inhibiting the DNMT1’s activity.3. ERa-negative breast cancer cell MDA-MB-435s re-sensitize to endocrine therapy after ERa re-expression in vitro and in vivo. It may therefore provide a novel therapeutic approach for ERa-negative breast cancer patients in clinical practice.