Evaluating the association of biallelic OGDHL variants with significant phenotypic heterogeneity.

BACKGROUND: Biallelic variants in OGDHL, encoding part of the α-ketoglutarate dehydrogenase complex, have been associated with highly heterogeneous neurological and neurodevelopmental disorders. However, the validity of this association remains to be confirmed. A second OGDHL patient cohort was recruited to carefully assess the gene-disease relationship. METHODS: Using an unbiased genotype-first approach, we screened large, multiethnic aggregated sequencing datasets worldwide for biallelic OGDHL variants. We used CRISPR/Cas9 to generate zebrafish knockouts of ogdhl, ogdh paralogs, and dhtkd1 to investigate functional relationships and impact during development. Functional complementation with patient variant transcripts was conducted to systematically assess protein functionality as a readout for pathogenicity. RESULTS: A cohort of 14 individuals from 12 unrelated families exhibited highly variable clinical phenotypes, with the majority of them presenting at least one additional variant, potentially accounting for a blended phenotype and complicating phenotypic understanding. We also uncovered extreme clinical heterogeneity and high allele frequencies, occasionally incompatible with a fully penetrant recessive disorder. Human cDNA of previously described and new variants were tested in an ogdhl zebrafish knockout model, adding functional evidence for variant reclassification. We disclosed evidence of hypomorphic alleles as well as a loss-of-function variant without deleterious effects in zebrafish variant testing also showing discordant familial segregation, challenging the relationship of OGDHL as a conventional Mendelian gene. Going further, we uncovered evidence for a complex compensatory relationship among OGDH, OGDHL, and DHTKD1 isoenzymes that are associated with neurodevelopmental disorders and exhibit complex transcriptional compensation patterns with partial functional redundancy. CONCLUSIONS: Based on the results of genetic, clinical, and functional studies, we formed three hypotheses in which to frame observations: biallelic OGDHL variants lead to a highly variable monogenic disorder, variants in OGDHL are following a complex pattern of inheritance, or they may not be causative at all. Our study further highlights the continuing challenges of assessing the validity of reported disease-gene associations and effects of variants identified in these genes. This is particularly more complicated in making genetic diagnoses based on identification of variants in genes presenting a highly heterogenous phenotype such as "OGDHL-related disorders".

Broadening the phenotypic and molecular spectrum of FINCA syndrome: Biallelic NHLRC2 variants in 15 novel individuals.

FINCA syndrome [MIM: 618278] is an autosomal recessive multisystem disorder characterized by fibrosis, neurodegeneration and cerebral angiomatosis. To date, 13 patients from nine families with biallelic NHLRC2 variants have been published. In all of them, the recurrent missense variant p.(Asp148Tyr) was detected on at least one allele. Common manifestations included lung or muscle fibrosis, respiratory distress, developmental delay, neuromuscular symptoms and seizures often followed by early death due to rapid disease progression.Here, we present 15 individuals from 12 families with an overlapping phenotype associated with nine novel NHLRC2 variants identified by exome analysis. All patients described here presented with moderate to severe global developmental delay and variable disease progression. Seizures, truncal hypotonia and movement disorders were frequently observed. Notably, we also present the first eight cases in which the recurrent p.(Asp148Tyr) variant was not detected in either homozygous or compound heterozygous state.We cloned and expressed all novel and most previously published non-truncating variants in HEK293-cells. From the results of these functional studies, we propose a potential genotype-phenotype correlation, with a greater reduction in protein expression being associated with a more severe phenotype.Taken together, our findings broaden the known phenotypic and molecular spectrum and emphasize that NHLRC2-related disease should be considered in patients presenting with intellectual disability, movement disorders, neuroregression and epilepsy with or without pulmonary involvement.

Corilagin prevents non-alcoholic fatty liver disease via improving lipid metabolism and glucose homeostasis in high fat diet-fed mice.

Non-alcoholic fatty liver disease (NAFLD) has been considered to be one of the most common chronic liver diseases. However, no validated pharmacological therapies have been officially proved in clinic due to its complex pathogenesis. The purpose of this study was to examine the protective effects of Corilagin (referred to Cori) against NAFLD in mice under a high fat diet (HFD) condition. Mice were fed either a normal control diet (NCD) or HFD with or without Cori (5 or 10 mg/kg body weight) for 15 weeks. In our results, Cori treatment significantly attenuated HFD-induced hepatic steatosis, high NAFLD activity score (NAD) and liver injury. Consistently, Cori treatment remarkably alleviated HFD-induced hepatic lipid accumulation (e.g., triglycerides (TG) and total cholesterol (TC) contents in liver), and improved plasma lipid concentrations (e.g., plasma TG, TC, low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c)). Moreover, Cori treatment ameliorated NAFLD associated metabolic disorders such as glucose intolerance and insulin resistance in HFD-fed mice. Additionally, Cori treatment dramatically changed HFD-induced liver gene expression profiles, and identified overlapped differentially expressed genes (DEGs) between NCD vs. HFD group and HFD vs. HCR (high fat diet plus treatment with Cori) group. With these DEGs, we observed a marked enrichment of Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, which were closely associated with the metabolic balance in liver. Particularly, we found several potential hub proteins against NAFLD development with analyses of protein-protein interaction (PPI) network and qPCR assays. Collectively, our results revealed the important protective effects of Cori against the progress of NAFLD, which was probably mediated through improving dysregulated lipid metabolism and insulin resistance in HFD-fed mice. Additionally, Cori-dependent overlapped DEGs might serve as a featured NAFLD-associated gene expression signature for the diagnosis, treatment, as well as drug discovery and development of NAFLD in the near future.

Multi source translation based projection completion for interior region of interest tomography with CBCT.

Interior tomography by rotary computed tomography (RCT) is an effective method to improve the detection efficiency and achieve high-resolution imaging for the region of interest (ROI) within a large-scale object. However, because only the X-rays through the ROI can be received by detector, the projection data is inevitably truncated, resulting in truncation artifacts in the reconstructed image. When the ROI is totally within the object, the solution of the problem is not unique, which is named interior problem. Fortunately, projection completion (PC) is an effective technique to solve the interior problem. In this study, we proposed a multi source translation CT based PC method (mSTCT-PC) to cope with the interior problem. Firstly, mSTCT-PC employs multi-source translation to sparsely obtain the global projection which covered the whole object. Secondly, the sparse global projection is utilized to fill up the truncated projection of ROI. The global projection and truncated projection are obtained under the same geometric parameters. Therefore, it omits the registration of projection. To verify the feasibility of this method, simulation and practical experiments were implemented. Compared with the results of ROI reconstructed by filtered back-projection (FBP), simultaneous iterative reconstruction technique-total variation (SIRT-TV) and the multi-resolution based method (mR-PC), the proposed mSTCT-PC is good at mitigating truncation artifacts, preserving details and improving the accuracy of ROI images.

Efficacy and safety of CDK4/6 inhibitors combined with endocrine therapy versus endocrine therapy alone in hormone receptor-positive, HER2-negative, advanced breast cancer: a systematic review and meta-analysis.

BACKGROUND: In previous studies on the application of cyclin-dependent kinase 4/6 (CDK4/6) inhibitors combined with endocrine therapy in advanced breast cancer, the outcomes of overall survival (OS) were inconsistent. This systematic review and meta-analysis aimed to further evaluate the clinical efficacy and safety of CDK4/6 inhibitors combined with endocrine therapy on patients with hormone receptor (HR)-positive and human epidermal growth factor receptor 2 (HER2)-negative advanced breast cancer. METHODS: Randomized controlled trials (RCTs) comparing CDK4/6 inhibitors plus endocrine therapy and endocrine therapy alone in patients with HR-positive and HER2-negative advanced breast cancer were searched in the databases of PubMed, Embase, Cochrane Library, China National Knowledge Infrastructure (CNKI), WANFANG and China Science and Technology Journal Database (VIP) up to November 2022. Hazard ratios (HRs) and confidence intervals (CI) of OS, progression-free survival (PFS), the time from randomization to the first recorded disease progression while the patient was receiving next-line therapy or death from any cause (PFS2), time to first subsequent chemotherapy after discontinuation (TTC), and objective response rate (ORR), clinical benefit rate (CBR), safety indicators were extracted. Stata 14.0 software was used for meta analysis and the Cochrane risk-of-bias tool 2.0 was used to evaluate the bias risk. RESULTS: A total of 9 RCTs with 4,920 participants were included. The addition of CDK4/6 inhibitors to endocrine therapy significantly prolonged OS (HR 0.76; 95% CI: 0.69-0.84; P<0.001), regardless of the application in first-line and second-line treatment, compared with endocrine therapy alone. Similar benefit was observed in PFS (HR 0.56; 95% CI: 0.52-0.60; P<0.001). Moreover, the CDK4/6 inhibitors group improved results of ORR [relative risk (RR) 1.43; 95% CI: 1.27-1.62; P<0.001], CBR (RR 1.24; 95% CI: 1.08-1.41; P<0.01 and RR 1.11; 95% CI: 1.06-1.18; P<0.001), PFS2 (HR 0.68; 95% CI: 0.60-0.76; P<0.001) and TTC (HR 0.65; 95% CI: 0.58-0.72; P<0.001). One of the included RCTs had performance bias. Publication bias was not significant. CONCLUSIONS: CDK4/6 inhibitors combined with endocrine therapy effectively prolong OS, PFS, PFS2, and TTC, and also improve ORR and CBR in patients with HR-positive, HER2-negative advanced breast cancer, and the safety was within the controllable range.

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.

Application of Periareolar Mammaplasty with the Tissue Folding Technique in Breast Reshaping following Polyacrylamide Hydrogel Removal.

BACKGROUND: Polyacrylamide hydrogel (PAAG) was used as an injectable implant for augmentation mammoplasty for over 30 years, but its use was ceased due to various related complications. The only way to treat these complications is PAAG removal, but this causes breast ptosis, nipple retraction, breast asymmetry, and skin laxity. OBJECTIVES: This article reports a new technique for breast reshaping after PAAG removal without prosthesis implantation. METHOD: From January 2015 to June 2018, twenty-three patients underwent periareolar mammoplasty with the tissue folding technique (PMTFT) for breast reshaping after PAAG removal. Postoperative breast shape and the degree of satisfaction of the patients were evaluated during follow-up. RESULTS: All patients recovered well without severe complications. All patients were satisfied with their postoperative breast shape and their symptoms were relieved after surgery. CONCLUSIONS: PMTFT provides satisfactory postoperative breast shape results. Economical, practical, and technical advantages were found over traditional prosthesis-mediated breast reconstruction. PMTFT can be an ideal surgical choice in appropriate cases.

Continuous postoperative negative pressure irrigation assisted mammaplasty in treating chronic refractory plasma cell mastitis.

BACKGROUND: Chronic refractory plasma cell mastitis (CRPCM) is an aseptic inflammation of the breast with a chronic course of the disease, extended treatment cycle (months to years), with a high recurrence rate. The objective of this study was to evaluate the efficacy of continuous postoperative negative pressure irrigation assisted mammaplasty (CPNPIAM) in treating CRPCM. METHODS: Between 2016 and 2018, 36 CRPCM patients receiving CPNPIAM were enrolled in this study. CPNPIAM mainly involved complete lesion removal, immediate breast mammaplasty, and continuous postoperative negative pressure irrigation. The age of the patients, local symptoms, history of treatment, the duration of the disease before surgery, hospitalization period, related risk factors, the success rate, the recurrence rate and patients' overall satisfaction ratings were analyzed in the article. RESULTS: Patients were aged between 22 and 53 years (mean 34.64 years). All patients had a history of conservative treatment or simple drainage. Local symptoms included inflammatory mass (n=36, 100%), abscess (n=33, 92%), nipple discharge (n=7, 19%), inflammatory plaque (n=34, 94%), and sinus tract formation (n=19, 53%). The lesion sizes ranged from 3 to 10 cm (mean 5.13 cm) in diameter. The mean hospitalization period was 8.42 days. The success rate was 100% (36/36) and the recurrence rate was 0% (0/36) at a 3-month follow-up. The patients' overall satisfaction ratings were "very good" (n=22, 61%), "good" (n=12, 33%), and "moderate" (n=2, 6%) with no poor or unsatisfactory ratings. CONCLUSIONS: CPNPIAM is an effective way of treating CRPCM, and showed a high success rate, a low recurrence rate, and high patient satisfaction.

Degradation of MCL-1 by bufalin reverses acquired resistance to osimertinib in EGFR-mutant lung cancer.

Although osimertinib, an EGFR tyrosine kinase inhibitor, has become the standard therapy for treating non-small cell lung cancer (NSCLC) patients with EGFR-activating mutation, upregulation of MCL-1 induces acquired resistance to osimertinib. Bufalin, a natural digoxin-like ingredient isolated from a traditional Chinese medicine Chan Su, has been shown to downregulate MCL-1 in NSCLC cells. However, whether bufalin reverses this acquired resistance to osimertinib in NSCLC cells remains unclear. In this study, bufalin reduced cell viability and promoted apoptosis in osimertinib-resistant cells. Moreover, co-treatment with bufalin and osimertinib restored the sensitivity of osimertinib-resistant cells to osimertinib-induced growth regression and apoptosis in vitro and in vivo. Mechanistically, MEK/ERK-dependent MCL-1 phosphorylation and Ku70-mediated MCL-1 overexpression confer osimertinib resistance in EGFR-mutant NSCLC cells. In osimertinib-resistant cells, bufalin modulates Ku70-mediated MCL-1 degradation, but not MEK/ERK/MCL-1 signaling. In conclusion, our study suggests that bufalin eliminates resistance to osimertinib by inhibiting Ku70-mediated MCL-1 overexpression, indicating that a combination of osimertinib and bufalin could be an effective additional treatment to overcome acquired resistance to osimertinib in NSCLC cells.

Corilagin Alleviates Nonalcoholic Fatty Liver Disease in High-Fat Diet-Induced C57BL/6 Mice by Ameliorating Oxidative Stress and Restoring Autophagic Flux.

Corilagin (Cori) possesses multiple biological activities. To determine whether Cori can exert protective effects against nonalcoholic fatty liver disease (NAFLD) and its potential mechanisms. C57BL/6 mice were fed with high-fat diet (HFD) alone or in combination with Cori (20 mg/kg, i.p.) and AML12 cells were exposed to 200 µM PA/OA with or without Cori (10 µM or 20 µM). Phenotypes and key indicators relevant to NAFLD were examined both in vivo and in vitro. In this study, Cori significantly ameliorated hepatic steatosis, confirmed by improved serum lipid profiles, and hepatic TC, TG contents, and the gene expression related to lipid metabolism in livers of HFD mice. Moreover, Cori attenuated HFD-mediated autophagy (including mitophagy) blockage by restoring autophagic flux, evidenced by increased number of autophagic double vesicles containing mitochondria, elevated LC3II protein levels, decreased p62 protein levels, as well as enhanced colocalization of autophagy-related protein (LC3, Parkin) and mitochondria. In accordance with this, Cori also reduced the accumulation of ROS and MDA levels, and enhanced the activities of antioxidative enzymes including SOD, GSH-Px, and CAT. In addition, Cori treatment improved mitochondrial dysfunction, evidenced by increased mitochondrial membrane potential (ΔΨm). In parallel with this, Cori decreased mitochondrial DNA oxidative damage, while increased mitochondrial biogenesis related transcription factors expression, mitochondrial DNA content and oxygen consumption rate (OCR). In conclusion, these results demonstrate that Cori is a potential candidate for the treatment of NAFLD via diminishing oxidative stress, restoring autophagic flux, as well as improving mitochondrial functions.

Interaction of positive coactivator 4 with histone 3.3 protein is essential for transcriptional activation of the luteinizing hormone receptor gene.

The luteinizing hormone receptor (LHR) is essential for sexual development and reproduction in mammals. We have established that Sp1 has a central role in derepression of LHR gene transcription induced by Trichostatin A (TSA) in MCF7 cells. Moreover, the co-activator PC4 which associates directly with Sp1 at the LHR promoter is essential for TSA-mediated LHR transcription. This study explores interactions of PC4 with histone proteins, which presumably triggers chromatin modifications during LHR transcriptional activation. TSA treatment of MCF7 cells expressing PC4-Flag protein induces acetylation of histone 3 (H3) and immunoprecipitation (IP) studies revealed its interaction with PC4-Flag protein. MS/MS analysis of the protein complex obtained after IP from TSA treated samples detected H3.3 acetylated at K9, K14, K18, K23 and K27 as a PC4 interacting protein. The association of PC4 with H3.3 was corroborated by IP and re-ChIP using H3.3 antibody. Similarly, IP and re-ChIP showed association of PC4 with H3 acetylated protein. Knockdown of PC4 in MCF7 cells reduced H3.3 enrichment, H3 acetylation at the Lys sites and LHR promoter activity in TSA treated cells despite an increase in H3 and H3.3 protein induced by TSA, linking PC4 to H3 acetylation and LHR transcription. Depletion of H3.3 A/B in MCF7 cells impair chromatin accessibility and enrichment of Pol II and TFIIB at the LHR promoter and its activation, resulting in marked reduction of LHR gene expression. Together, these findings point to the critical role of PC4 and its association with acetylated H3.3 in TSA-induced LHR gene transcription.

Efficacy of a Chinese Herbal Medicine Compound Zhangpi Ointment against Hydroxyurea-Induced Leg Ulcers: A Prospective, Randomized, Open-Label, Controlled Clinical Trial.

Objective. The randomized controlled trial was to evaluate the efficacy of topical Chinese herbal Zhangpi Ointment for hydroxyurea-induced leg ulcers in patients with myeloproliferative neoplasms. Patients and Methods. This single-center, prospective, randomized, open-label, controlled clinical trial conducted at Shanghai Ninth People's Hospital enrolled 54 patients with hydroxyurea-induced leg ulcers. Patients were randomly assigned to the control group (n = 27) treated with chlorhexidine dressing or the intervention group (n = 27) treated with the Zhangpi Ointment. Finally, 26 patients in the control group and 23 patients in the intervention group completed 8 weeks of observation. Results. The rate of complete healing was 100% for the intervention group, which was significantly higher than that of the control group (96.15%) (P<0.05). Furthermore, the intervention group achieved a significantly higher rate of wound healing (95.56%) than the control group (69.02%) at week 4 (P<0.01). The intervention group took 34 ± 5 days to achieve complete healing while the control group took 41 ± 7 days (P < 0.01). Moreover, grade 3/4 side effects were observed in neither group. Conclusion. The Zhangpi Ointment is effective in promoting the healing of hydroxyurea-induced leg ulcers in patients with myeloproliferative neoplasms, providing a therapeutic option for a condition that is recalcitrant to conventional therapy.

Curcumin suppresses Notch­1 signaling: Improvements in fatty liver and insulin resistance in rats.

Curcumin is a well­known phenolic substance and has many pharmacological effects associated with metabolism. However, the exact molecular mechanisms underlying this process have yet to be determined. The Notch pathway is a signal transduction pathway involved in energy metabolism. The present study aimed to investigate the effects of curcumin administration on glucose­lipid metabolism in rats subjected to a high fat diet, and investigate changes in Notch­1 signaling. Sprague­Dawley rats (n=40) were randomly divided into four groups (10 rats/group): Control diet group, high fat diet group, high fat diet plus curcumin low dose group and high fat diet plus curcumin high dose group. Following 8 weeks of treatment with curcumin (100 mg/kg in the low dose group and 200 mg/kg in the high dose group), serum metabolic markers and hepatic gene expression patterns were investigated. No differences in body weight following 8 weeks of curcumin administration (P>0.05) were observed; however, curcumin treatment did reduce visceral fat levels (peri­epididymal and peri­renal), and decreased cholesterol, triglyceride and low­density lipoprotein levels in serum compared with the high fat diet rats that did not receive curcumin (P<0.05, P<0.01). An oral glucose tolerance test and an intraperitoneal insulin tolerance test revealed that insulin resistance was reduced (P<0.05 or P<0.01) and tissue section analysis revealed that hepatosteatosis was attenuated following treatment with curcumin. Furthermore, the protein expression of Notch­1 and its downstream target Hes­1 were suppressed. These effects were also in parallel with an upregulation of fatty acid oxidation­associated gene expression, including peroxisome proliferator­activated receptor (PPAR)­α, carnitine palmitoyltransferase 1 and PPAR­Î³ (P<0.05). In addition, curcumin administration led to a downregulation in the expression of lipogenic genes, including sterol regulatory element­binding protein, fatty acid synthase and acetyl­CoA carboxylase (P<0.05). The expression of inflammation­associated genes, including nuclear factor­κB, tumor necrosis factor­α and prostaglandin­endoperoxide synthase 2 were also suppressed. The results of the present study suggest that the hepatic Notch­1 pathway can be suppressed via curcumin treatment, which may ameliorate fatty liver and insulin resistance in rats subjected to a high fat diet.

Comparison of breast-conserving surgery with mastectomy in locally advanced breast cancer after good response to neoadjuvant chemotherapy: A PRISMA-compliant systematic review and meta-analysis.

BACKGROUND: The application of breast-conserving surgery (BCS) on patients with locally advanced breast cancer (LABC) with good response to neoadjuvant chemotherapy (NACT) still remains controversial. The objective in this study is to analyze the safety of BCS in the management of LABC in patients with good response to NACT. METHODS: We searched the electronic databases of Medline (Pubmed) and Cochrane Library for reports on local recurrence (LR), regional recurrence (RR), distant recurrence (DR), 5-year disease-free survival (DFS) or 5-year overall survival (OS) in patients with LABC receiving BCS or mastectomy (MT) and with good response to NACT. Based on the research results, we conducted a meta-analysis using Review Manager 5.3. RESULTS: Our study showed that 16 studies with a combined total of 3531 patients, of whom 1465 patients underwent BCS, whereas 2066 patients underwent MT. There was no significant heterogeneity among these studies (Q statistic: P = .88; I = 0%). Patients with good response to NACT showed no significant difference in LR and RR [odd ratio (OR) = 0.83; 95% confidence interval (CI): 0.60-1.15; P = .26; OR = 0.56; 95% CI: 0.33-0.93; P = .03], while we figured out a lower DR (OR = 0.51; 95% CI: 0.42-0.63; P < .01), a higher DFS (OR = 2.35; 95% CI: 1.84 to 3.01, P < .01) and a higher OS (OR = 2.12; 95% CI: 1.51 to 2.98, P < .01) in BCS compared with MT. CONCLUSION: This meta-analysis concluded that BCS was a safe surgery for patients with LABC and had good response to NACT.

Yiqi formula enhances the antitumor effects of erlotinib for treatment of triple-negative breast cancer xenografts.

Yiqi formula (YF), a traditional herbal prescription, has long been used to treat triple-negative breast cancer (TNBC) patients. The present study aims to investigate the effects and the related mechanism of YF for treatment of TNBC xenografts. MDA-MB-231 (human TNBC) cells were subcutaneously injected into the second mammary fat pad of 40 female nude mice, which were divided into four groups: control, erlotinib (an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor), YF, and combination (YF plus erlotinib). All treatments were administered orally for 30 days. Inhibition rate of tumor weight by erlotinib, YF, and the combination was 26.47%, 17.24%, and 39.15%, respectively. Western blotting showed that YF, erlotinib, and the combination downregulated p-EGFR (P < 0.01) and p-Akt1 (pT308) (P < 0.05) and upregulated PTEN compared with control, and the combination was more efficacious than erlotinib alone (P < 0.05). Similar results were detected by immunohistochemistry. Real-time quantitative PCR showed that YF, erlotinib, and the combination increased PTEN mRNA (P < 0.05, P < 0.01) compared with control, and the combination was more efficacious than erlotinib alone (P < 0.05). In conclusion, YF can regulate the main components of the PI3K/Akt pathway in TNBC xenografts. When YF was used in combination with erlotinib, it enhanced the antitumor effects of erlotinib on TNBC xenografts. These findings suggest that YF is suitable to use for the treatment of TNBC patients.

Bufalin Reverses HGF-Induced Resistance to EGFR-TKIs in EGFR Mutant Lung Cancer Cells via Blockage of Met/PI3k/Akt Pathway and Induction of Apoptosis.

The epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib, have shown promising therapeutic efficacy in nonsmall cell lung cancer (NSCLC) patients harboring epidermal growth factor receptor- (EGFR-) activating mutation. However, the inevitable recurrence resulting from acquired resistance has limited the clinical improvement in therapy outcomes. Many studies demonstrate that hepatocyte growth factor- (HGF-) Met axis plays an important role in tumor progression and drug sensitivity. HGF may induce resistance to EGFR-TKIs in EGFR mutant lung cancer cells by Met/PI3K/Akt signaling. The purpose of this study was to determine whether bufalin, a major bioactive component of Venenum Bufonis, could reverse HGF-induced resistance to reversible and irreversible EGFR-TKIs in mutant lung cancer cells PC-9, HCC827, and H1975. Our studies showed that bufalin could reverse resistance to reversible and irreversible EGFR-TKIs induced by exogenous HGF in EGFR mutant lung cancer cells by inhibiting the Met/PI3K/Akt pathway and inducing death signaling. These results suggested that bufalin might have a potential to overcome HGF-induced resistance to molecular-targeted drugs for lung cancer.

[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.

Coactivator function of positive cofactor 4 (PC4) in Sp1-directed luteinizing hormone receptor (LHR) gene transcription.

The LHR has an essential role in sexual development and reproductive function, and its transcription is subjected to several modes of regulation. In this study, we investigated PC4 coactivator function in the control of LHR transcription. Knockdown of PC4 by siRNA inhibited the LHR basal promoter activity and trichostatin A (TSA)-induced gene transcriptional activation and expression in MCF-7 cells. While overexpression of PC4 alone had no effect on the LHR gene, it significantly enhanced Sp1- but not Sp3-mediated LHR transcriptional activity. PC4 directly interacts with Sp1 at the LHR promoter, and this interaction is negatively regulated by PC4 phosphorylation. The coactivator domain (22-91 aa) of PC4 and DNA binding domain of Sp1 are essential for PC4/Sp1 interaction. ChIP assay revealed significant occupancy of PC4 at the LHR promoter that increased upon TSA treatment. Disruption of PC4 expression significantly reduced TSA-induced recruitment of TFIIB and RNAP II, at the promoter. PC4 functions are beyond TSA-induced phosphatase release, PI3K-mediated Sp1 phosphorylation, and HDAC1/2/mSin3A co-repressor release indicating its role as linker coactivator of Sp1 and the transcriptional machinery. These findings demonstrated a critical aspect of LHR modulation whereby PC4 acts as a coactivator of Sp1 to contribute to the human of LHR transcription.

Participation of signaling pathways in the derepression of luteinizing hormone receptor transcription.

The luteinizing hormone receptor (LHR) transcription is subject to an epigenetic regulatory mode whereby the proximal Sp1 site acts as an anchor to recruit histone deacetylases (HDAC)1/2 and the Sin3A co-repressor complex. This results in promoter-localized histone hypo-acetylation that contributes to the silencing of LHR transcriptional expression. Chromatin changes resulting from site-specific acetylation and methylation of histones regulate LHR gene expression. The HDAC inhibitor TSA-induced cell-specific phosphatase release from the promoter, which serves as an 'on' mechanism for Sp1 phosphorylation by phosphatidylinositol 3-kinase/protein kinase Czeta (PI3K/PKCzeta) at Ser641, leading to p107 repressor derecruitment and LHR transcriptional activation. The methylation status of the promoter provides another layer of modulation in a cell-specific manner. Maximal derepression of the LHR gene is dependent on complete DNA demethylation of the promoter in conjunction with histone hyperacetylation and release of repressors (p107 and HDAC/Sin3A). Independently, the PKC-alpha/Erk pathway, participates in LHR gene expression through induction of Sp1 phosphorylation at Ser site(s) other than Ser641. This causes dissociation of the HDAC1/mSin3A from the promoter, recruitment of TFIIB and Pol II, and transcriptional activation. Collectively, these findings demonstrate that LHR gene expression at the transcriptional level is regulated by complex and diverse networks, in which coordination and interactions between these regulatory effectors are crucial for silencing/activation of LHR expression.

Unlocking repression of the human luteinizing hormone receptor gene by trichostatin A-induced cell-specific phosphatase release.

Our previous studies demonstrated that the histone deacetylase inhibitor, trichostatin A (TSA), induces derepression of the human luteinizing hormone receptor (LHR) gene by de-recruitment of the pRB homologue p107 repressor from the promoter in JAR and MCF-7 cancer cells. TSA initiates a mechanism whereby the phosphatidylinositol 3-kinase/protein kinase zeta (PKCzeta) cascade phosphorylates Sp1 at Ser-641, which is essential for the release of the repression of LHR transcription. The present studies have revealed that dissociation of serine/threonine protein phosphatases PP2A and PP1 from the LHR promoter mediates TSA-induced activation of LHR gene transcription in a cell-specific manner. Changes in chromatin structure induced by TSA cause the release of PP2A in JAR cells or of PP1 in MCF-7 cells, which is associated with Sp1 directly or through histone deacetylase 1/2, respectively, at the promoter. This favors the phosphorylation of Sp1 mediated by the phosphatidylinositol 3-kinase/PKCzeta pathway, which in turn causes the release of the p107 inhibitor from Sp1 and marked transcriptional activation of the LHR. These findings reveal the importance of phosphatases in the control of LHR transcription, where the balance between phosphatidylinositol 3-kinase/PKCzeta and phosphatases could be critical for up- and down-regulation of LHR gene expression in physiological and pathological settings.