Alterations in Serum Lipids and Lipoproteins Induced by Neoadjuvant Chemotherapy in Patients with Osteosarcoma around the Knee Joint: A Retrospective Analysis.

OBJECTIVE: To investigate the serum lipid profiles of patients with localized osteosarcoma around the knee joint before and after neoadjuvant chemotherapy. METHODS: After retrospectively screening the data of 742 patients between January 2007 and July 2020, 50 patients aged 13 to 39 years with Enneking stage II disease were included in the study. Serum lipid levels, including total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), lipoprotein-α [Lp(a)], and apolipoprotein A1, B, and E (ApoA1, ApoB, and ApoE), and clinicopathological characteristics were collected before and after neoadjuvant chemotherapy. RESULTS: The mean levels of TC, TG, and ApoB were significantly increased following neoadjuvant chemotherapy (16%, 38%, and 20%, respectively, vs. pretreatment values; P<0.01). The mean levels of LDL-C and ApoE were also 19% and 16% higher, respectively (P<0.05). No correlation was found between the pretreatment lipid profile and the histologic response to chemotherapy. An increase in Lp(a) was strongly correlated with the Ki-67 index (R=0.31, P=0.023). Moreover, a trend toward longer disease-free survival (DFS) was observed in patients with decreased TG and increased LDL-C following chemotherapy, although this difference was not statistically significant (P=0.23 and P=0.24, respectively). CONCLUSION: Significant elevations in serum lipids were observed after neoadjuvant chemotherapy in patients with localized osteosarcoma. There was no prognostic significance of pretreatment serum lipid levels on histologic response to neoadjuvant chemotherapy. The scale of increase in serum Lp(a) might have a potential prognostic role in osteosarcoma. Patients with increased LDL-C or reduced TG after chemotherapy seem to exhibit a trend toward favorable DFS.

Analysis of human Ki-67 gene promoter and identification of the Sp1 binding sites for Ki-67 transcription.

Ki-67 as a cell proliferation marker is tightly associated with maintenance and regulation of the cell division. To understand the mechanism of Ki-67 gene expression and regulation, we first cloned 5'-flanking region and identified the Ki-67 core promoter. The deletion analysis and the dual luciferase reporter assay were used to locate the Ki-67 core promoter from -223 to +12 nt relative to the transcriptional initiation site, which is the TATA less, GC rich region comprised of several putative Sp1 binding sites. Compared with the hTERT promoter and Survivin promoter, the Ki-67 core promoter possessed higher transcription activity and more desirable tumor selectivity. In order to further demonstrate the contribution of transcription factor Sp1 on regulating the Ki-67 gene transcription, we confirmed three Sp1 binding sites from -170 to -145 nt, from -63 to -38 nt, and from -14 to +12 nt existed in the Ki-67 core promoter by the supershift assay. The deletion mutagenesis, together with the dual luciferase reporter assay, indicated that these Sp1 binding sites, particularly the region from -170 to -145 nt, were involved in positive regulation of the Ki-67 gene expression. Collectively, it was demonstrated that the region from -223 to +12 nt could drive the transcription of the Ki-67 gene, and the Sp1 binding site is essential to transcriptional regulation of the Ki-67 gene.

A critical role of Sp1 transcription factor in regulating the human Ki-67 gene expression.

Ki-67 plays a crucial role in cell proliferation as well as maintenance or regulation of cell division. The mechanism governing the Ki-67 gene expression remains unknown. Thus, we cloned the core promoter of the human Ki-67 gene and further investigated its transcriptional regulation. The putative Sp1 binding sites were confirmed by electrophoretic mobility shift assay together with an anti-Sp1 antibody-mediated supershift assay. Deletion mutagenesis and firefly luciferase reporter gene assay demonstrated the essential contribution of Sp1 on transcriptional activation of the Ki-67 gene. In this study, we first confirm that there are three Sp1 binding sites in the Ki-67 core promoter. Two Sp1 sites (one at position -159 to -145 nt and the other at position -14 to +12 nt) are mainly involved in transcriptional regulation of the Ki-67 gene. Overexpression of Sp1 can enhance the Ki-67 promoter activity. However, down-regulation of Sp1 expression using siRNA-Sp1 and mithramycin effectively inhibits the Ki-67 gene transcription. Our results suggest that Sp1 is essential for basal promoter activity of the human Ki-67 gene. Inhibition of the Ki-67 transcriptional activity through abolishment of Sp1 may provide the useful prospect for gene therapy.

p53 regulates Ki-67 promoter activity through p53- and Sp1-dependent manner in HeLa cells.

The expression of the human Ki-67 protein, which is strictly associated with cell proliferation, is regulated by a variety of cellular mediators. In this study, we studied the effects of p53 on Ki-67 promoter in HeLa cells using luciferase reporter assay. The results showed that: (1) p53 inhibited Ki-67 promoter activity in a dose-dependent manner, (2) the p53-binding motifs mediated part of the transcriptional repression of Ki-67 promoter through a sequence-specific interaction with p53, (3) p53 was able to repress the Sp1-stimulated Ki-67 promoter activity, and (4) the Sp1-binding sites were responsible for the p53-mediated transcriptional repression of Ki-67 promoter. In conclusion, p53 inhibited Ki-67 promoter activity via p53- and Sp1-dependent pathways, and the interaction between p53 and Sp1 might be involved in the transcriptional regulatory mechanisms.

The effect of methylated oligonucleotide targeting Ki-67 gene in human 786-0 renal carcinoma cells.

To investigate the effect of methylated oligonucleotide (MON) targeting Ki-67 promoter on the expression of Ki-67 gene and the proliferation and apoptosis of the human 786-0 renal carcinoma cells, human 786-0 cells were transfected with MON. The activity of Ki-67 promoter was detected by dual-luciferase reporter assay system. Among the five methylated oligonucleotides (MON(1)-MON(5)), MON(4) is the best excellent one in the inhibition of the Ki-67 promoter activity. The activity of Ki-67 promoter is decreased to 77.88% in 40-nM group, 50.07% in 80-nM group, 35.63% in 120-nM group, 26.09% in 160-nM group, and 16.98% in 200-nM group compared with 0-nM group. The activity of Ki-67 promoter in MON group is decreased to 61.96% at 8 h, 48.93% at 12 h, 15.97% at 24 h, 26.00% at 36 h, 35.01% at 48 h, 46.08% at 72 h, and 66.12% at 96 h compared with pGLBK235 group. These results show that the effect of MON is time- and dose-dependent. The activity of Ki-67 in MON group is decreased to 16.73% compared with pGLBK235 group, while the control groups have no significant difference. The expression of Ki-67 gene in 786-0 cells was detected by RT-PCR and immunohistochemistry, respectively. The expression of Ki-67 mRNA is decreased to 61.04% and that of Ki-67 protein is decreased to 32.07% in MON group compared with the blank group. The proliferation of 786-0 cells was determined by WST-8. The cell proliferation in MON group is decreased to 61.02% at 24 h, 73.78% at 48 h, 79.72% at 72 h, and 91.53% at 96 h compared with the blank group. The cell apoptosis was measured by annexin V and propidium iodide. The number of apoptosis cells in MON group is 2.42 times of that in the blank group at earlier period and 2.57 times at mid-anaphase. We detected the effect of MON on the expression of bax and p53 by Western blot. Compared with the blank group, the expression of bax protein in MON group is increased by 66.12%, while the expression of p53 is decreased to 67.31%. Our study demonstrates that the methylated oligonucleotide targeting Ki-67 promoter has a remarkable effect on the inhibition of Ki-67 expression and the proliferation of the human 786-0 renal carcinoma cells and can induce apoptosis of the 786-0 cells.

Reciprocal Role Of DNA Methylation And Sp1 Binding In Ki-67 Gene Transcription.

PURPOSE: DNA methylation plays major regulatory roles in gene transcription. Our previous studies confirmed that Ki-67 promoter is hypomethylated and Sp1 is a transcriptional activator of Ki-67 gene in cancer cells. However, whether Sp1-mediated transcriptional activation of Ki-67 is related to its methylation has not been studied yet. MATERIALS AND METHODS: In this study, we confirmed that methylated CpG binding protein 2 (MBD2) binding to methylated DNA hindered the binding of Sp1 to Ki-67 promoter and then repressed Ki-67 transcription through chromatin immunoprecipitation (ChIP) and quantitative real-time PCR (qRT-PCR). Co-immunoprecipitation (Co-IP), ChIP, methylation-specific PCR (MS-PCR) and Western blot were utilized to analyze the effects of Sp1 binding to Ki-67 promoter on its methylation status. RESULTS: Less DNA methyltransferase 1 (DNMT1) bound to the Ki-67 promoter in MKN45 cells than in HK-2 cells. Histone acetyltransferase p300 that was recruited by Sp1 to Ki-67 promoter could attenuate the methylation level of Ki-67 promoter. Furthermore, higher expression of Sp1 and Ki-67 was related to the overall survival (OS), first progression (FP) and post-progression survival (PPS) in gastric cancer by scrutinizing bioinformatics datasets. CONCLUSION: Taken together, our findings suggested that hypomethylation of Ki-67 promoter enhanced the binding of Sp1, which in turn maintained hypomethylation of promoter, leading to increase Ki-67 expression in cancer cells. Sp1 and Ki-67 could act promising prognostic biomarkers for clinical diagnosis and treatment of cancer.

Effect of radiotherapy on survival of women with locally excised ductal carcinoma in situ of the breast: a Surveillance, Epidemiology, and End Results population-based analysis.

BACKGROUND: Although it has been previously reported that radiotherapy (RT) effectively reduced the incidence of local recurrence of ductal carcinoma in situ (DCIS) following breast-conserving surgery (BCS), little is known about the effect of RT on survival of patients with locally excised DCIS. PATIENTS AND METHODS: Using Surveillance, Epidemiology, and End Results registry data, we selected 56,968 female DCIS patients treated with BCS between 1998 and 2007. Overall survival (OS) and breast cancer-specific survival (BCSS) were compared among patients who received RT or no RT using the Kaplan-Meier methods and Cox proportional hazards regression models. RESULTS: Median follow-up was 91 months. In the multivariable model, patients receiving postoperative RT had better OS than those undergoing BCS alone (hazard ratio [HR] 0.59, 95% confidence interval [CI] 0.53-0.67, P<0.001). This pattern remained after stratification by estrogen receptor (ER) status and age. In contrast, RT delivery was not significantly associated with improved BCSS (HR 0.71, 95% CI 0.48-1.03, P=0.073). However, after stratifying by the above two variables, RT contributed to better BCSS in ER-negative/borderline patients (HR 0.41, 95% CI 0.19-0.88, P=0.023) and younger patients (≤50 years old; HR 0.37, 95% CI 0.15-0.91, P=0.030). CONCLUSION: Our analysis confirms the beneficial effect of RT on OS in women with locally excised DCIS and reveals the specific protective effect of RT on BCSS in ER-negative/borderline and younger patients.

An elevated peripheral blood lymphocyte-to-monocyte ratio predicts favorable response and prognosis in locally advanced breast cancer following neoadjuvant chemotherapy.

PURPOSE: Neoadjuvant chemotherapy (NCT) is a standard treatment option for locally advanced breast cancer. However, the lack of an efficient method to predict treatment response and patient prognosis hampers the clinical evaluation of patient eligibility for NCT. An elevated lymphocyte-to-monocyte ratio (LMR) has been reported to be associated with a favorable prognosis for certain hematologic malignancies and for nasopharyngeal carcinoma; however, this association has not been investigated in breast cancer. The purpose of this study was to evaluate whether pre-NCT LMR analysis could predict the prognosis of patients with locally advanced breast cancer. METHODS: A retrospective cohort of 542 locally advanced breast cancer patients (T3/T4 and/or N2/N3 disease) receiving NCT followed by radical surgery was recruited between May 2002 and August 2011 at the Fudan University Shanghai Cancer Center. Counts for pre-NCT peripheral absolute lymphocytes and monocytes were obtained and used to calculate the LMR. RESULTS: Univariate and multivariate analysis revealed that higher LMR levels (≥4.25) were significantly associated with favorable DFS (P = 0.009 and P = 0.011, respectively). Additionally, univariate analysis revealed that a higher lymphocyte count (≥1.5×109/L) showed borderline significance for improved DFS (P = 0.054), while a lower monocyte count (<0.4×109/L) was associated with a significantly better DFS (P = 0.010). CONCLUSIONS: An elevated pre-NCT peripheral LMR level was a significantly favorable factor for locally advanced breast cancer patient prognosis. This easily obtained variable may serve as a valuable marker to predict the outcomes of locally advanced breast cancer.