Paradoxical Mitophagy Regulation by PINK1 and TUFm.

Aberrant mitophagy has been implicated in a broad spectrum of disorders. PINK1, Parkin, and ubiquitin have pivotal roles in priming mitophagy. However, the entire regulatory landscape and the precise control mechanisms of mitophagy remain to be elucidated. Here, we uncover fundamental mitophagy regulation involving PINK1 and a non-canonical role of the mitochondrial Tu translation elongation factor (TUFm). The mitochondrion-cytosol dual-localized TUFm interacts with PINK1 biochemically and genetically, which is an evolutionarily conserved Parkin-independent route toward mitophagy. A PINK1-dependent TUFm phosphoswitch at Ser222 determines conversion from activating to suppressing mitophagy. PINK1 modulates differential translocation of TUFm because p-S222-TUFm is restricted predominantly to the cytosol, where it inhibits mitophagy by impeding Atg5-Atg12 formation. The self-antagonizing feature of PINK1/TUFm is critical for the robustness of mitophagy regulation, achieved by the unique kinetic parameters of p-S222-TUFm, p-S65-ubiquitin, and their common kinase PINK1. Our findings provide new mechanistic insights into mitophagy and mitophagy-associated disorders.

ETS1 is associated with cisplatin resistance through IKKα/NF-κB pathway in cell line MDA-MB-231.

BACKGROUND: Platinum-based drugs are used extensively in neoadjuvant chemotherapy for triple-negative breast cancer (TNBC), but their use can be limited by resistance. In this study, we established cisplatin (DDP) resistant TNBC cells to investigate the potential relationship among ETS1, IKKα/NF-κB and resistance. METHODS: The sensitivity was evaluated by MTT, apoptosis analysis. The intracellular DDP concentration difference was tested by inductively coupled plasma mass spectrometry (ICP-MS) method. Molecular pathological mechanism of DDP resistance was explored by microarray analysis and PPI network analysis. The ETS1, NF-κB signaling change were assessed by western blot and q-PCR in vitro and vivo. The existing binds between ETS1 and the core IKKα promoter were found by luciferase assay and chromatin immunoprecipitation technique (ChIP). RESULTS: MDA-MB-231/DDP (231/DDP) cell had a higher IC50 value of cisplatin, lower intracellular DDP concentration, and lower apoptosis ratio than MDA-MB-231 (231/wt) cell line treated with DDP. Increased ABC transporters were induced by the activation of NF-κB pathway in 231/DDP cells. ETS1, RPL6, RBBP8, BIRC2, PIK3A and RARS were six important genes for DDP-resistance based on PPI network and expression validation. Protein expression of ETS1 and IKKα were significantly up-regulated in 231/DDP cells. However, inhibition of ETS1 expression enhances chemo-sensitivity to DDP and reversed the activation of NF-κB pathway in 231/DDP cells and subcutaneous transplantation tumor in vivo. Moreover, there is existing binds between ETS1 and the core IKKα promoter though luciferase assay and ChIP. CONCLUSION: This study enables us to understand the functions of ETS1 in TNBC chemotherapy and suggests that ETS1 could be used as a novel marker of poor response to DDP and a potential therapeutic target for TNBC chemotherapy.

[External therapy of plasma cell mastitis by jiuyi powder using partial least-squares discriminant analysis: a safety analysis].

OBJECTIVE: To evaluate the safety and the clinical value of external use of jiuyi Powder (JP) in treating plasma cell mastitis using partial least-squares discriminant analysis (PLSDA). METHODS: Totally 50 patients with plasma cell mastitis treated by external use of JP were observed and biochemical examinations of blood and urine detected before application, at day 4 after application, at day 1 and 14 after discontinuation. Blood mercury and urinary mercury were detected before application, at day 1, 4, and 7 after application, at day 1 and 14 after discontinuation. Urinary mercury was also detected at 28 after discontinuation and 3 months after discontinuation. The information of wound, days of external application and the total dosage of external application were recorded before application, at day 1, 4, and 7 after application, as well as at day 1 after discontinuation. Then a discriminant model covering potential safety factors was set up by PLSDA after screening safety indices with important effects. The applicability of the model was assessed using area under ROC curve. Potential safety factors were assessed using variable importance in the projection (VIP). RESULTS: Urinary ß2-microglobulin (ß2-MG), urinary N-acetyl-ß-D-glucosaminidase (NAG), 24 h urinary protein, and urinary α1-microglobulin (α1-MG) were greatly affected by external use of JP in treating plasma cell mastitis. The accuracy rate of PLSDA discriminate model was 74. 00%. The sensitivity, specificity, and the area under ROC curve was 0. 7826, 0. 7037, and 0. 8084, respectively. Three factors with greater effect on the potential safety were screened as follows: pre-application volume of the sore cavity, days of external application, and the total dosage of external application. CONCLUSIONS: PLSDA method could be used in analyzing bioinformation of clinical Chinese medicine. Urinary ß2-MG and urinary NAG were two main safety monitoring indices. Days of external application and the total dosage of external application were main factors influencing blood mercury and urine mercury. A safety classification simulation model of treating plasma cell mastitis by external therapy of JP was established by the two factors, which could be used to assess the safety of external application of JP to some extent.

Shenqi Fuzheng injection reverses M2 macrophage-mediated cisplatin resistance through the PI3K pathway in breast cancer.

BACKGROUND: Shenqi Fuzheng injection (SQFZ) combined with chemotherapy can sensitize tumour cells. However, the mechanisms underlying SQFZ's effects remain unknown. In human breast cancer cell lines and M2 macrophages, we showed that SQFZ was a significantly potent agent of sensitization. METHODS: The human breast cancer cell line, MDA-MB-231/DDP, and the human acute leukaemia mononuclear cell line, THP-1, were used. MDA-MB-231/DDP breast cancer xenografts were established to monitor tumour growth. Resistance-associated proteins were examined by western blotting. Levels of cytokines and chemokines were detected by ELISA. Cell viability was measured using the MTT assay. Apoptosis was detected by flow cytometric analysis. RESULTS: SQFZ significantly enhanced the capability of cisplatin to reduce tumour mass. SQFZ and cisplatin decreased the expression of CD206 by 1.89-fold and increased that of CD86 by 1.76-fold as compared to cisplatin alone. The levels of PGE2, IL-6, and CCL1 decreased significantly, and the activation of p-PI3K and the expressions of P-gp and ABCG2 were also inhibited by SQFZ in combination with cisplatin treatment in vivo. The survival following cisplatin administration of 60 µM and 120 µM reduced significantly in the presence of SQFZ in MDA-MB-231/DDP and M2 co-cultured cells. IGF-1, a PI3K activator, combined with SQFZ weakened the effects of SQFZ-induced apoptosis from 28.7% to 10.5%. The effects of IGF-1 on increasing the expressions of P-gp, ABCG2, and Bcl-2, and decreasing that of Bax were reversed by SQFZ. CONCLUSION: Our findings provide evidence that SQFZ is a potential therapeutic drug for cancer therapy.

Differences and significance of peripheral blood interleukin-6 expression between patients with granulomatous lobular mastitis and those with benign breast tumors.

Objective: It is unclear whether the mechanism of the interleukin (IL)-6 signaling pathway is similar between granulomatous lobular mastitis (GLM) and benign breast tumors. This study aimed to explore the differences and significance of peripheral blood IL-6 and related cytokines, routine blood test results, and C-reactive protein (CRP) levels between patients with GLM and benign breast tumors. Methods: Seventy-three inpatients with GLM who underwent surgery and 60 patients with benign breast tumors diagnosed based on pathological findings between November 2022 and May 2023 were included. The white blood cell (WBC) and neutrophil (NEU) counts were determined using an automatic blood cell analyzer, the CRP level was determined by an immunoturbidimetric assay, and serum IL-6 and related cytokine levels were determined by an enzyme-linked immunosorbent assay. Results: The WBC, NEU, and CRP values in patients with GLM were significantly higher than those in patients with benign breast tumors (P < 0.01). Serum IL-6 levels were significantly higher in patients with GLM than in those with benign breast tumors (P < 0.01). There were no significant differences in the serum concentrations of IL-1ß, IL-7, and interferon (IFN)-γ between patients with GLM and those with benign breast tumors (P > 0.05), but the tumor necrosis factor (TNF)-α level was higher in patients with GLM than in those with benign breast tumors (P < 0.01). In patients with GLM, the Pearson correlation analysis showed that the IL-6 level was positively correlated with NEU, NEU%, CRP, IL-17, and TNF-α values (P < 0.01). Additionally, the IL-6 level was weakly positively correlated with WBC and IFN-γ values. Conversely, in patients with benign breast tumors, the IL-6 level was not significantly correlated with the aforementioned indicators in routine blood tests but was positively correlated with IL-17, IFN-γ, and TNF-α values (P < 0.01). Conclusions: IL-6, NEU, NEU%, and CRP values were significantly elevated in patients with GLM compared to those with benign breast tumors, indicating that IL-6 plays an important role in the development and onset of GLM. The correlation between these cytokines and the development and progression of benign breast tumors needs to be further explored, as cytokines such as IL-6 may provide effective markers for the treatment of GLM.

Association between endocrine therapy and cognitive decline in breast cancer based on propensity score matching.

Purpose: To study the status quo of the cognitive function of the breast cancer patients with (who went through) the endocrine therapy by the epidemiological investigation, analyze the key factor of the cognition impairment and explore the impact of the endocrine therapy time on the cognition decline after using Propensity Score Matching to balance the covariates. Methods: In this study, the epidemiological questionnaire information was collected from 226 female breast cancer endocrine treatment patients who visited the Breast Clinic of Longhua Hospital Affiliated to Shanghai University of Chinese Medicine from November 2020 to February 2022, and the results of the overall cognitive function, the function test of each cognitive domain, the patient's self-cognition, quality of life, and emotional status evaluation of the patients. In this study, according to the principle of random matching, the nearest matching method with a matching tolerance of 0.2 and a matching ratio of 1:2 was used for orientation score matching. After the covariant such as age, BMI, and duration of education were balanced, the effects of the duration of endocrine therapy on the overall cognitive function and the functions of each cognitive domain were analyzed. Results: In 226 cases of female breast cancer patients (who went through) the endocrine therapy, the propensity score matching was performed, ultimately, 99 were ruled out, successful matched ones were 49 of the cognition-decline group and 78 of the standard group. With age, education time, BMI and other covariates balanced, the endocrine therapy duration was the risk factor of the cognition impairment (P < 0.05, OR = 1.296, 95% CI = 1.008-1.665), with the extension of endocrine treatment time, there was a rising risk of the cognition impairment (LLA statistic = 5.872, P < 0.05). The cognitive domain scores in the cognition-decline group were lower than the standard group (P < 0.05), but there was a difference in self-report cognition. Conclusion: The endocrine therapy duration was the risk factor for the cognition impairment of the breast cancer patients, and with prolonged endocrine treatment, there was a rising (an increasing) risk for the cognition impairment.

Construction and validation of a risk prediction model for aromatase inhibitor-associated bone loss.

Purpose: To establish a high-risk prediction model for aromatase inhibitor-associated bone loss (AIBL) in patients with hormone receptor-positive breast cancer. Methods: The study included breast cancer patients who received aromatase inhibitor (AI) treatment. Univariate analysis was performed to identify risk factors associated with AIBL. The dataset was randomly divided into a training set (70%) and a test set (30%). The identified risk factors were used to construct a prediction model using the eXtreme gradient boosting (XGBoost) machine learning method. Logistic regression and least absolute shrinkage and selection operator (LASSO) regression methods were used for comparison. The area under the receiver operating characteristic curve (AUC) was used to evaluate the performance of the model in the test dataset. Results: A total of 113 subjects were included in the study. Duration of breast cancer, duration of aromatase inhibitor therapy, hip fracture index, major osteoporotic fracture index, prolactin (PRL), and osteocalcin (OC) were found to be independent risk factors for AIBL (p < 0.05). The XGBoost model had a higher AUC compared to the logistic model and LASSO model (0.761 vs. 0.716, 0.691). Conclusion: The XGBoost model outperformed the logistic and LASSO models in predicting the occurrence of AIBL in patients with hormone receptor-positive breast cancer receiving aromatase inhibitors.

[Overview of studies on detoxification effect of smilacis glabrae rhizoma on mercury poisoning].

Mercury-containing preparations are widely used in surgery department of traditional Chinese medicine and have made remarkable achievements. But they are toxic to human kidney, nerve, immune, etc. Smilacis Glabrae Rhizoma is sweet, tasteless and neutral in nature and able to enter liver and stomach channels and detoxify mercury poisoning. This article summarizes the mercury poisoning and the detoxification effect of Smilacis Glabrae Rhizoma in ancient records, pharmaceutical studies and clinical application, in order to provide ideas and methods for the safe use of mercury-containing preparations in surgery department of traditional Chinese medicine.

[Effects of external use of jiuyi dan for one month on blood and urine mercury levels and liver and kidney functions of rabbits].

OBJECTIVE: To observe the changes of the blood and urine mercury (Hg) levels and liver & kidney functions of rabbits after administration of Jiuyi Dan (calcined gypsum-Sheng Dan 9: 1) for 1 month and the recovery of rabbits after the drug withdrawal. METHOD: The rabbits were randomly divided into 2 groups: the calcined gypsum group and the Jiuyi Dan group. After 36 mg of calcined gypsum and 40 mg of Jiuyi Dan were used on the surface of wound (5 cm x 5 cm) on one side of rabbit back for 4 h, the surfaces of wound were washed by saline. The bloods were taken from the rabbit hearts before and after the drug administration for 14 and 28 days, and after the drug withdrawal for 7, 40, 71, and 92 days for determining Hg level in blood, and liver & kidney function indicators (ALT, AST, CREAT and BUN). The Hg level in urine collected from bladders was examined while rabbits were dissected after the drug withdrawal for 1, 40, 71, and 92 days. RESULT: The Hg level in blood was significantly increased (P < 0.01) after the rabbits were administrated with drugs for 14 and 28 days and after the drug treatment was stopped for 7 and 40 days. The Hg level in urine was significantly enhanced after the drug withdrawal for 1, 40, 71 days. However, the liver & kidney indicators were not influenced. CONCLUSION: The Hg level in rabbit blood and urine was significantly increased after the consecutive administration of double-dose Jiuyi Dan for 1 month. However, the blood Hg level and urine Hg level recover after the drug withdrawal for 71 days and 3 months, respectively. The liver & kidney indicators do not significantly change with the dose.

[Effects of Astragalus polysaccharide on proliferation and Akt phosphorylation of the basal-like breast cancer cell line].

OBJECTIVE: To investigate the effects of Astragalus polysaccharide (APS) on proliferation of basal-like breast cancer cell line MDA-MB-468 cells and Akt phosphorylation in MDA-MB-468 cells. METHODS: APS at different concentrations was used to culture MDA-MB-468 cells for different time periods, and then proliferation of MDA-MB-468 cells was assayed using methyl thiazolyl tetrazolium (MTT) assay to determine the time- and dose-dependent effects of APS. For observing the effects of APS on phosphor-Akt (p-Akt), in-cell Western blot method was used after 1, 2, 4 and 7 d of culture in APS to detect protein expressions of p-Akt (Thr308) and p-Akt (Ser473). Protein levels of the key targets in p53/murine double minute 2 (MDM2) signaling pathway, such as p53, MDM2 and phosphatase and tensin homolog deleted on chromosome ten (PTEN) were also detected. After PTEN gene was silenced by small interfering RNA (siRNA) in MDA-MB-468 cells, expressions of p-Akt (Thr308 and Ser473) were assayed by the in-cell Western blot method after 2 d of APS treatment. RESULTS: APS at 1 and 0.5 mg/mL concentrations effectively inhibited the proliferation of MDA-MB-468 cells and was used in subsequent tests. Compared with the control group, APS decreased the protein expression of p-Akt (Thr308) in MDA-MB-468 cells after 1-, 2-, 4- and 7-day culture, and also decreased the protein expression of p-Akt (Ser473) and up-regulated the protein expression of MDM2 in MDA-MB-468 cells after 1- and 2-day culture. Expressions of p53 and PTEN were up-regulated after 7 d of APS culture. After silencing PTEN gene by siRNA, APS could not mediate Akt phosphorylation. CONCLUSION: APS can inhibit proliferation of basal-like breast cancer cell line MDA-MB-468, and down-regulate the expression of Akt phosphorylation. The antiproliferation mechanisms may be related to its effects of up-regulating the expressions of p53 and PTEN by regulating p53/MDM2 positive and negative feedback loops.

[Study protocol for a self-controlled case study to evaluate the safety and standardization for external application of Chinese medicine Jiuyi Powder].

BACKGROUND: As the main medicinal powder for drawing out pus and removing necrotic tissue in external therapies of traditional Chinese surgery, Sheng Powder has made great contributions to the treatment of inflammatory wounds and has the unique bactericidal and decay-discharging function that can not be replaced by antibiotics. However, Sheng Powder has toxicity because it contains mercury. So far, there is no clinical research on the standards of dose and usage of Sheng Powder and there is a lack of objective and quantitative criteria for operating standards and monitoring of toxicity and side effects. Therefore, the authors choose Jiuyi Powder, one of the most commonly used Sheng Powder, to evaluate the safety of its external use, and form a standardization program for clinical implementation. METHODS AND DESIGN: This study is a nonrandomized, nonblinded, self-controlled case study. There will be two key stages in the study. In stage one, 10 patients with plasma cell mastitis will be enrolled. The patients will receive continuous external application of Jiuyi Powder with a fixed dose (160 mg/d). Blood mercury and urine mercury levels will be dynamically tested at different time intervals, and the observation of pharmacokinetic parameters will be conducted after Jiuyi Powder has been absorbed by the surface of the wound. In stage two, 30 patients with each of the four conditions including postoperative wounds of head or facial surgeries, plasma cell mastitis, anal fistula and chronic absorption ulcer of the shank will be enrolled, respectively. According to the dose of 1.5 mg/cm(2), Jiuyi Powder will be externally applied to the wound surface for 14 d. On the basis of the first-phase study, the test time will be chosen and the toxicity outcome will be detected to evaluate the safety of external application of Jiuyi Powder and to establish recommendations for standardized clinical use. DISCUSSION: The purpose of this study is to evaluate the safety of the external application of Jiuyi Powder through a two-stage study. The pharmacokinetic parameters of external application of Jiuyi Powder at the clinical dose range, the changes in blood and urinary mercury levels and related safety indexes will be observed on different wounds located in different positions externally. The evaluation of whether Jiuyi Powder could be a routine medication for drawing out pus and removing necrotic tissue in inflammatory and refractory wounds will be stated. TRIAL REGISTRATION NUMBER: ChiCTR-TNC-11001366.

Qigong for women with breast cancer: An updated systematic review and meta-analysis.

OBJECTIVE: The purpose of this review was to evaluate the effectiveness of Qigong in improving the quality of life and relieving fatigue, sleep disturbance, and cancer-related emotional disturbances (distress, depression, and anxiety) in women with breast cancer. METHODS: The PubMed, Cochrane Central Register of Controlled Trials, Web of Science, Sinomed, Wanfang, VIP, and China National Knowledge Infrastructure databases were searched from their inceptions to March 2020 for controlled clinical trials. Two reviewers selected relevant trials that assessed the benefit of Qigong for breast cancer patients independently. A methodological quality assessment was conducted according to the criteria of the 12 Cochrane Back Review Group for risk of bias independently. A meta-analysis was performed by Review Manager 5.3. RESULTS: This review consisted of 17 trials, in which 1236 cases were enrolled. The quality of the included trials was generally low, as only five of them were rated high quality. The results showed significant effectiveness of Qigong on quality of life (n = 950, standardized mean difference (SMD), 0.65, 95 % confidence interval (CI) 0.23-1.08, P =  0.002). Depression (n = 540, SMD = -0.32, 95 % CI -0.59 to -0.04, P =  0.02) and anxiety (n = 439, SMD = -0.71, 95 % CI -1.32 to -0.10, P =  0.02) were also significantly relieved in the Qigong group. There was no significant benefit on fatigue (n = 401, SMD = -0.32, 95 % CI  0.71 to 0.07, P = 0.11) or sleep disturbance relief compared to that observed in the control group (n = 298, SMD = -0.11, 95 % CI  0.74 to 0.52, P = 0.73). CONCLUSION: This review shows that Qigong is beneficial for improving quality of lifeand relieving depression and anxiety; thus, Qigong should be encouraged in women with breast cancer.

Formononetin relieves the facilitating effect of lncRNA AFAP1-AS1-miR-195/miR-545 axis on progression and chemo-resistance of triple-negative breast cancer.

This investigation attempted to discern whether formononetin restrained progression of triple-negative breast cancer (TNBC) by blocking lncRNA AFAP1-AS1-miR-195/miR-545 axis. We prepared TNBC cell lines (i.e. MDA-MB-231 and BT-549) and normal human mammary epithelial cell line (i.e. MCF-10A) in advance, and the TNBC cell lines were, respectively, transfected by pcDNA3.1-lncRNA AFAP1-AS1, si-lncRNA AFAP1-AS1, pcDNA6.2/GW/EmGFP-miR-545 or pcDNA6.2/GW/EmGFP-miR-195. Resistance of TNBC cells in response to 5-Fu, adriamycin, paclitaxel and cisplatin was evaluated through MTT assay, while potentials of TNBC cells in proliferation, migration and invasion were assessed via CCK8 assay and Transwell assay. Consequently, silencing of lncRNA AFAP1-AS1 impaired chemo-resistance, proliferation, migration and invasion of TNBC cells (P<0.05), and over-expression of miR-195 and miR-545, which were sponged and down-regulated by lncRNA AFAP1-AS1 (P<0.05), significantly reversed the promoting effect of pcDNA3.1-lncRNA AFAP1-AS1 on proliferation, migration, invasion and chemo-resistance of TNBC cells (P<0.05). Furthermore, CDK4 and Raf-1, essential biomarkers of TNBC progression, were, respectively, subjected to target and down-regulation of miR-545 and miR-195 (P<0.05), and they were promoted by pcDNA3.1-lncRNA AFAP1-AS1 at protein and mRNA levels (P<0.05). Additionally, formononetin significantly decreased expressions of lncRNA AFAP1-AS1, CDK4 and Raf-1, while raised miR-195 and miR-545 expressions in TNBC cells (P<0.05), and exposure to it dramatically contained malignant behaviors of TNBC cells (P<0.05). In conclusion, formononetin alleviated TNBC malignancy by suppressing lncRNA AFAP1-AS1-miR-195/miR-545 axis, suggesting that molecular targets combined with traditional Chinese medicine could yield significant clinical benefits in TNBC.

Attenuation of epigenetic regulator SMARCA4 and ERK-ETS signaling suppresses aging-related dopaminergic degeneration.

How complex interactions of genetic, environmental factors and aging jointly contribute to dopaminergic degeneration in Parkinson's disease (PD) is largely unclear. Here, we applied frequent gene co-expression analysis on human patient substantia nigra-specific microarray datasets to identify potential novel disease-related genes. In vivo Drosophila studies validated two of 32 candidate genes, a chromatin-remodeling factor SMARCA4 and a biliverdin reductase BLVRA. Inhibition of SMARCA4 was able to prevent aging-dependent dopaminergic degeneration not only caused by overexpression of BLVRA but also in four most common Drosophila PD models. Furthermore, down-regulation of SMARCA4 specifically in the dopaminergic neurons prevented shortening of life span caused by α-synuclein and LRRK2. Mechanistically, aberrant SMARCA4 and BLVRA converged on elevated ERK-ETS activity, attenuation of which by either genetic or pharmacological manipulation effectively suppressed dopaminergic degeneration in Drosophila in vivo. Down-regulation of SMARCA4 or drug inhibition of MEK/ERK also mitigated mitochondrial defects in PINK1 (a PD-associated gene)-deficient human cells. Our findings underscore the important role of epigenetic regulators and implicate a common signaling axis for therapeutic intervention in normal aging and a broad range of age-related disorders including PD.

Cisplatin-resistant MDA-MB-231 Cell-derived Exosomes Increase the Resistance of Recipient Cells in an Exosomal miR-423-5p-dependent Manner.

BACKGROUND: Chemoresistance blunts the therapeutic effect of cisplatin (DDP) on Triple-Negative Breast Cancer (TNBC). Researchers have not determined to date whether exosomes confer DDP resistance to other breast cancer cells or whether exosomal transfer of miRNAs derived from DDP-resistant TNBC cells confer DDP resistance. OBJECTIVE: The aim of this study was to investigate the role of exosomes in chemoresistance in breast cancer. METHODS: MDA-MB-231 cells resistant to DDP (231/DDP) were established. Exosomes were isolated from 231/DDP cells (DDP/EXO) and characterized by measuring the levels of protein markers, nanoparticle tracking analysis and transmission electron microscopy. MDA-MB-231, MCF-7 and SKBR-3 cell lines were treated with the isolated DDP/EXOs and cell proliferation and cytotoxicity to DDP were evaluated using MTT assays and apoptosis analyses. Western blotting was used to examine P-glycoprotein (P-gp) expression. Additionally, a microarray was used to analyse microRNA (miRNA) expression profiles in MDA-MB-231 and 231/DDP exosomes. The effects on miRNAs were determined using RT-PCR. Exosomal miR-423-5p was extracted, and differential expression was verified. The MTT cell viability assay, flow cytometry, and Transwell and immunofluorescence assays were performed to determine if differential expression of miR-423-5p sensitized cells to DDP in vitro. RESULTS: Under a transmission electron microscope, the isolated exosomes exhibited a round or oval shape with a diameter ranging between 40 and 100 nm. DDP/EXOs labelled with PKH67 were taken up by MDA-MB-231 cells. After an incubation with DDP/EXOs, the cell lines exhibited a higher IC50 value for cisplatin, P-gp expression, migration and invasion capabilities and a lower apoptosis rate. Furthermore, 60 miRNAs from exosomes derived from 231/DDP cells were significantly up-regulated compared to exosomes from MDA-MB-231 cells. Notably, compared to the corresponding sensitive exosomes, miR-370-3p, miR-423-5p and miR-373 were the most differentially expressed miRNAs in DDP-resistant exosomes. We chose miR-423-5p, and up-regulation and down-regulation of exosomal miR-423-5p expression significantly affected DDP resistance. CONCLUSIONS: Exosomes from DDP-resistant TNBC cells (231/DDP) altered the sensitivity of other breast cancer cells to DDP in an exosomal miR-423-5p dependent manner. Our research helps to elucidate the mechanism of DDP resistance in breast cancer.

Long noncoding RNA HCP5 contributes to cisplatin resistance in human triple-negative breast cancer via regulation of PTEN expression.

BACKGROUND: Abnormally expressed long non-coding RNA (lncRNA) is associated with the development of breast cancer and multidrug resistance. However, the molecular mechanism by which lncRNA regulates cisplatin (DDP) in triple-negative breast cancer (TNBC) remains unclear. METHODS: DDP resistant cell line MDA-MB-231/DDP was established by gradually increasing doses of DDP. Abnormal expression of lncRNA between MDA-MB-231 and MDA-MB-231/DDP was evaluated with microarray. In addition, cell proliferation was evaluated by CCK-8 and Ki67 assays. Furthermore, cell apoptosis was evaluated by cell apoptosis and TUNEL assays. Western blotting assay was used to detect the protein expression. In vivo animal study was performed finally. RESULTS: HCP5 were significantly upregulated in MDA-MB-231/DDP cells compared with MDA-MB-231 cells. Overexpression of HCP5 promoted DDP resistance in MDA-MB-231 cells by inhibiting PTEN expression. In contrast, inhibition of HCP5 reversed DDP resistance in MDA-MB-231/ DDP cells by upregulating PTEN. In vivo experiments confirmed that downregulation of HCP5 inhibited DDP resistance in TNBC xenograft. CONCLUSION: We found that overexpression of HCP5 contributed to DDP resistance in TNBC, while downregulation of HCP5 reversed the resistance via upregulating PTEN level. Therefore, DDP combined with downregulation of HCP5 might be considered as a therapeutic approach for the treatment of DDP-resistant TNBC.

[Effects of Astragalus injection on proliferation of basal-like breast cancer cell line MDA-MB-468].

OBJECTIVE: To investigate the effects of Astragalus injection (AI) on basal-like breast cancer cell line MDA-MB-468 and murine bone marrow stromal stem cells (mMSCs). METHODS: MDA-MB-468 cells and primary cultured mMSCs were treated by different concentrations of AI, and with untreated MDA-MB-468 cells as blank control. The morphology of cells was observed by phase-contrast inverted microscope and transmission electron microscopy. Cytotoxic effects of AI on MDA-MB-468 cells and mMSCs were evaluated by cell counting kit-8 (CCK-8) assay. Cell cycle and apoptosis of MDA-MB-468 cells induced by AI were measured by flow cytometry. Lactate dehydrogenase (LDH) activity in supernatants was measured by enzymatic colorimetric method. The expressions of epidermal growth factor receptor (EGFR) and p53 protein in MDA-MB-468 cells were evaluated by streptavidin-biotin-peroxidase complex method. RESULTS: A time-dependent cytotoxic effect of 1 g/ml AI was observed in MDA-MB-468 cells. 1 g/ml AI also had cytotoxic effect on mMSCs, but its effect was not better than cisplatin. 0.1 g/ml AI could promote the proliferation of mMSCs. Different concentrations of AI could all induce the apoptosis of MDA-MB-468 cells. There was no significant difference in LDH activity in the supernatants between blank control group and AI-treated and cisplatin-treated groups. AI could down-regulate the expressions of EGFR and p53 protein. CONCLUSION: The effects of AI on MDA-MB-468 cells and mMSCs are related to the concentration of AI, and its mechanism of inhibiting the proliferation of MDA-MB-468 cells may be due to down-regulation of the expressions of EGFR and p53 protein.

[Effects of Chinese herbal medicine for warming kidney on proliferation of normal human mammary epithelial cells in primary culture].

OBJECTIVE: To evaluate the effects of Chinese herbal medicine for warming kidney on proliferation of normal human mammary epithelial cells in primary culture. METHODS: The normal human mammary epithelial cells were dissociated by digestion with collagenase type I. The morphological identification of normal human mammary epithelial cells in primary culture was determined under inverted phase contrast microscope and transmission electron microscope. The estradiol and progesterone were added to culture medium to induce the proliferation of normal human mammary epithelial cells in primary culture, and the different doses of Chinese herbal medicine for warming kidney and tamoxifen were also added into the culture medium. The MTT assay was used to evaluate the proliferation of normal human mammary epithelial cells in primary culture under the different conditions of interventions. RESULTS: The normal human mammary epithelial cells in primary culture presented typically morphological features of normal human mammary epithelial cells in vitro. The proliferation of normal human mammary epithelial cells in primary culture was significantly improved by intervention of estradiol and progesterone both with concentration of 1x10(-5) g/L. The high-dose Chinese herbal medicine for warming kidney could obviously promote the proliferation of normal human mammary epithelial cells in primary culture, while the proliferation was obviously inhibited by low-dose Chinese herbal medicine for warming kidney. The tamoxifen exerted no effects on the proliferation of normal human mammary epithelial cells in primary culture. CONCLUSION: The estradiol and progesterone can enhance the ability of proliferation of normal human mammary epithelial cells in primary culture. The regulating effect of Chinese herbal medicine for warming kidney on proliferation of normal human mammary epithelial cells in primary culture is dose-dependent.