Synthesis, isolation, characterization of C3-C11 bridge-bond isomer of paclitaxel and its antitumor effect via inducing A549 cells pyroptosis.

During the chemical manufacturing control processing of new paclitaxel formulations, a photodegradation impurity called C3-C11 bridge-bond isomer appeared. Our work describes the synthesis, isolation, purification, and structural characterization methods using four spectroscopies: FT-IR, UV, NMR (1H and 13 C), and LC-MS. In addition, we discovered that the C3-C11 bridge-bond isomer can promote A549 cells pyroptosis, and increase pyroptosis-related proteins, including cleaved-caspase 3, cleaved-PARP, GSDME-N, and lactate dehydrogenase, thus making it anti-tumor effects. The study offered data suggesting that the C3-C11 bridge bond isomer may be used as an anti-tumour drug in the future.

The influence of blood sample processing on blood-based DNA methylation signatures.

Stability is crucial for the clinical applicable biomarker such as DNA methylation profiles. However, the influence of various blood processing on the DNA methylation signatures have been barely studied. Here, we systematically evaluated the impact of temporary storage and frozen and thaw on the levels of DNA methylation. The methylation intensities of 13 CpG loci from 53 individuals (42 healthy participants and 11 lung cancer patients) whose blood samples were processed by up to 14 various protocols were quantitatively determined by the mass spectrometry, obtaining more than 8,000 quantitative data. We disclosed a trend of accumulatively increased DNA methylation along with time when the blood from healthy people were stored for up to 96 h at room temperature (RT), whereas the storage at 4°C had much weaker effects or no impact. On the contrary, the methylation patterns in the blood of lung cancer patients were more stable at both RT and 4°C even for 96 h. Multiple cycles of frozen and thaw could result in demethylation, but was more significant to the blood than to the extracted DNA. Our study indicated that the blood processing in vitro could influence the DNA methylation signatures and introduce unexpected biases.

The Association Between Breast Cancer and Blood-Based Methylation of CD160, ISYNA1 and RAD51B in the Chinese Population.

Recent studies have identified DNA methylation signatures in the white blood cells as potential biomarkers for breast cancer (BC) in the European population. Here, we investigated the association between BC and blood-based methylation of cluster of differentiation 160 (CD160), inositol-3-phosphate synthase 1 (ISYNA1) and RAD51 paralog B (RAD51B) genes in the Chinese population. Peripheral blood samples were collected from two independent case-control studies with a total of 272 sporadic early-stage BC cases (76.5% at stage I&II) and 272 cancer-free female controls. Mass spectrometry was applied to quantitatively measure the levels of DNA methylation. The logistic regression and non-parametric tests were used for the statistical analyses. In contrast to the protective effects reported in European women, we reported the blood-based hypomethylation in CD160, ISYNA1 and RAD51B as risk factors for BC in the Chinese population (CD160_CpG_3, CD160_CpG_4/cg20975414, ISYNA1_CpG_2, RAD51B_CpG_3 and RAD51B_CpG_4; odds ratios (ORs) per -10% methylation ranging from 1.08 to 1.67, p < 0.05 for all). Moreover, hypomethylation of CD160, ISYNA1 and RAD51B was significantly correlated with age, BC subtypes including estrogen receptor (ER)-negative BC tumors, triple negative tumors, BC cases with larger size, advanced stages and more lymph node involvement. Our results supported the report in European women that BC is associated with altered methylation of CD160, ISYNA1 and RAD51B in the peripheral blood, although the effects are opposite in the Chinese population. The difference between the two populations may be due to variant genetic background or life styles, implicating that the validations of epigenetic biomarkers in variant ethnic groups are warranted.

RPTOR methylation in the peripheral blood and breast cancer in the Chinese population.

BACKGROUND: Altered regulatory-associated protein of mTOR, complex 1 (RPTOR) methylation levels in peripheral blood was originally discovered as breast cancer (BC)-associated risk factor in Caucasians. OBJECTIVE: To explore the relationship between RPTOR methylation and BC in the Chinese population, we conducted two independent case-control studies. METHODS: Peripheral blood samples were collected from a total of 333 sporadic BC cases and 378 healthy female controls for the DNA extraction and bisulfite-specific PCR amplification. Mass spectrometry was applied to quantitatively measure the levels of methylation. The logistic regression, Spearman's rank correlation, and Non-parametric tests were used for the statistical analyses. RESULTS: In our study, we found an association between BC and RPTOR_CpG_4 hypomethylation in the general population (per-10% of methylation, OR 1.29, P = 0.012), and a weak association between BC and RPTOR_CpG_8 hypomethylation in the women with older age (per-10% of methylation, OR 2.34, P = 0.006). We also identified age as a confounder for the change of RPTOR methylation patterns, especially at RPTOR_CpG_4, which represented differential methylation comparing age groups especially in the BC cases (age < 50 years vs age ≥ 50 years by Mann-Whitney U test, P < 0.0001 for BC cases and P = 0.079 for controls). CONCLUSION: Our study validated the association between hypomethylation of RPTOR and BC risk in the Chinese population also with weak effect and mostly for postmenopausal women. In addition, our findings provided novel insight for the regulation of DNA methylation upon aging or the change of hormone levels.

The association between RAPSN methylation in peripheral blood and breast cancer in the Chinese population.

DNA methylation in peripheral blood is associated with breast cancer (BC) but has mainly been studied in Caucasian populations. We investigated the association between blood-based methylation of receptor-associated protein of the synapse (RAPSN) and BC in Chinese population. The methylation levels of 12 RAPSN CpG sites were quantitatively evaluated by mass spectrometry in two case-control studies with 283 sporadic BC cases and 331 controls totally. The association was analyzed by logistic regression adjusted for covariants. The RAPSN methylation levels in patients with variant clinical characteristics were investigated by non-parametric tests. We found a significant association between BC and altered RAPSN methylation in blood in women at premenopausal and perimenopausal (age < 50 years old), but not in the elder women. This was approved by two independent case-control studies as well as by combining the subjects of the two studies (taken all subjects together, age < 50 years old, per 5% of methylation, odds ratio (OR) range from 1.17 to 1.30 for two CpG sites; OR = 0.75 for one CpG site; all p values < 0.02). This age-related RAPSN methylation was further modified by human epidermal growth factor receptor 2 (HER2) status (age < 50 years old, HER2 negative, per 5% of methylation, OR range from 1.27 to 1.48 for two CpG sites; OR = 0.76 for one CpG site; all p values < 0.02). We elucidated an association between BC and blood-based RAPSN methylation influenced by age and the status of HER2 in Chinese population.

The Association Between Breast Cancer and Blood-Based Methylation of S100P and HYAL2 in the Chinese Population.

Previous work has shown that DNA methylation in peripheral blood may be associated with malignancy; however, these studies have mainly been conducted within Caucasian populations. Here, we investigated the association between blood-based methylation of S100 calcium-binding protein P gene (S100P) and hyaluronoglucosaminidase 2 gene (HYAL2) and breast cancer (BC) via mass spectrometry in two independent case-control studies of the Chinese population with a total of 351 BC cases and 427 cancer-free female controls. In Study I, in which subjects had an average of 45 years, hypomethylation of S100P showed a protective effect for women ≤45 years (six out of nine CpG sites, p < 0.05) but not for women >45 years. In contrast, hypomethylation of HAYL2 was not correlated with BC in women ≤45 years but was a risk factor for women >45 years (three out of four CpG sites, p < 0.05). We proposed an age-dependent correlation between BC and methylation of S100P and HYAL2 and performed further validation in Study II with older subjects (average age = 52.5 years), where hypomethylation of both S100P and HYAL2 was a risk factor for BC (p < 0.05 for 10 CpG sites) as reported in Caucasians who develop BC around 55 years old. Together with the observation that Chinese cancer-free females having variant basal methylation levels comparing to Caucasians, we assumed that blood-based methylation might be modified by ethnic background, hormone status, and lifestyle. Here, we highlighted that the epigenetic biomarkers warrant validations when its application in variant ethnic groups is considered.