Proximity labeling proteomics reveals Kv1.3 potassium channel immune interactors in microglia.

Microglia are resident immune cells of the brain and regulate its inflammatory state. In neurodegenerative diseases, microglia transition from a homeostatic state to a state referred to as disease associated microglia (DAM). DAM express higher levels of proinflammatory signaling molecules, like STAT1 and TLR2, and show transitions in mitochondrial activity toward a more glycolytic response. Inhibition of Kv1.3 decreases the proinflammatory signature of DAM, though how Kv1.3 influences the response is unknown. Our goal was to identify the potential proteins interacting with Kv1.3 during transition to DAM. We utilized TurboID, a biotin ligase, fused to Kv1.3 to evaluate potential interacting proteins with Kv1.3 via mass spectrometry in BV-2 microglia following TLR4-mediated activation. Electrophysiology, western blotting, and flow cytometry were used to evaluate Kv1.3 channel presence and TurboID biotinylation activity. We hypothesized that Kv1.3 contains domain-specific interactors that vary during a TLR4-induced inflammatory response, some of which are dependent on the PDZ-binding domain on the C-terminus. We determined that the N-terminus of Kv1.3 is responsible for trafficking Kv1.3 to the cell surface and mitochondria (e.g. NUDC, TIMM50). Whereas, the C-terminus interacts with immune signaling proteins in an LPS-induced inflammatory response (e.g. STAT1, TLR2, and C3). There are 70 proteins that rely on the C-terminal PDZ-binding domain to interact with Kv1.3 (e.g. ND3, Snx3, and Sun1). Furthermore, we used Kv1.3 blockade to verify functional coupling between Kv1.3 and interferon-mediated STAT1 activation. Overall, we highlight that the Kv1.3 potassium channel functions beyond conducting the outward flux of potassium ions in an inflammatory context and that Kv1.3 modulates the activity of key immune signaling proteins, such as STAT1 and C3.

Most total pancreatectomies for ductal adenocarcinoma potentially can Be replaced by whipple over the splenic artery: a before and after study.

INTRODUCTION: Recently, more and more total pancreatectomy (TP) has been performed for central-located pancreatic ductal cell adenocarcinoma (PDCA) which abuts or involves both gastroduodenal and splenic arteries and demands transaction of both of them for a complete resection. Spiked by Warshaw's procedure (spleen-preserving distal pancreatectomy with excision of splenic vessels), we developed a new procedure "Whipple over the splenic artery (WOTSA)" to replace TP by leftward extension of pancreatic parenchyma transaction line and preservation of pancreatic tail and spleen after excision of splenic artery. This uncontrolled before and after study assesses the safety and efficacy of a new technique "Whipple over the splenic artery (WOTSA)" as a treatment for PDAC which traditionally requires total pancreatectomy (TP) for a complete excision. METHODS: The study group comprised 40 consecutive patients who underwent WOTSA for PDAC between August 2019 and September 2022. Their clinicopathological characteristics and survival were compared with those of a historical control group comprising 30 consecutive patients who underwent TP between January 2016 and July 2019. RESULTS: None of the 40 patients in the WOTSA group required reoperation due to infarction of the pancreas and/or spleen remnant. DM medication after WOTSA were none in 19, oral hypoglycemic agents in 19, and insulin preparations in 2 patients. Compared with TP, patients who underwent WOTSA exhibited similar rates of major operative complications, clear pancreatic parenchyma transaction margin, and number of harvested positive lymph nodes, but higher rate of adjuvant chemotherapy completion and a trend toward better median disease free survival (14 vs. 7.5 mo, P=0.023). CONCLUSIONS: Compared to TP, WOTSA can be safely performed and have much better postoperative glycemic status without cost of higher operative risk or impaired surgical radicality. These findings indicate that most TPs for PDAC potentially can be replaced by WOTSAs.

Novel Non-duct-to-Mucosa Pancreaticojejunostomy Reconstruction After Pancreaticoduodenectomy: Focus on the Occurrence of Post-pancreatectomy Hemorrhage and Intra-abdominal Abscess.

BACKGROUND: Postoperative pancreatic fistulas (POPFs) are considered inevitable in some patients after pancreaticoduodenectomy (PD), and measures to minimize their clinical impact are needed. Postpancreatectomy hemorrhage (PPH) and intra-abdominal abscess (IAA) are the most severe POPF-related complications, and concomitant leakage of contaminated intestinal content is considered the main cause. An innovative method, modified non-duct-to-mucosa pancreaticojejunostomy (TPJ), was created to prevent concomitant leakage of intestinal content, and its effectiveness was compared between two periods. METHODS: All PD patients undergoing pancreaticojejunostomy from 2012 to 2021 were included. The TPJ group consisted of 529 patients recruited from January 2018 to December 2021. A total of 535 patients receiving the conventional method (CPJ) from January 2012 to June 2017 were used as a control group. PPH and POPF were defined according to the International Study Group of Pancreatic Surgery definition, but only PPH grade C was included for analysis. An IAA was defined as a collection of postoperative fluid managed by CT-guided drainage with documental culture. RESULTS: There were no significant differences in the rate of POPF between the two groups (46.0% vs. 44.8%; p = 0.700). Furthermore, the percentages of bile in the drainage fluid in the TPJ and CPJ groups were 2.3% and 9.2%, respectively (p < 0.001). Lower proportions of PPH (0.9% vs. 6.5%; p < 0.001) and IAA (5.7% vs. 10.8%; p < 0.001) were observed for TPJ than for CPJ. On adjusted models, TPJ was significantly associated with a lower rate of PPH (odds ratio [OR] 0.132, 95% confidence interval [CI] 0.051-0.343; p < 0.001) and IAA (OR 0.514, 95% CI 0.349-0.758; p = 0.001) than CPJ. CONCLUSIONS: TPJ is feasible to be performed and is associated with a similar rate of POPF but a lower percentage of concomitant bile in the drainage fluid and subsequent rates of PPH and IAA than CPJ.

Solid Pseudopapillary Neoplasms of the Pancreas Across Races Demonstrate Disparities with Comparably Good Prognosis.

BACKGROUND: Solid pseudopapillary neoplasms (SPNs) of the pancreas are rare with low-grade malignancy and unclarified clinicopathological features. This study aimed to examine their characteristics and re-evaluate current treatments. METHODS: Databases from three sources were screened for patients with SPNs. We compared the perioperative variables, clinical data, overall survival (OS), and prognostic factors for recurrence among the three corresponding cohorts. RESULTS: We identified 286 patients diagnosed with SPNs between 1988 and 2020. Patients were mostly women (81%; median age: 38 years), and peak incidence was observed in women of 20-29 years of age. SPNs had a peak incidence in Asian men at 50-59 years of age (p = 0.002) and a delayed peak incidence in Asian women at 30-39 years of age (p < 0.001). Treatment strategies differed significantly across the institutions and included variations in the number of harvested lymph nodes and rates of vascular resection. Lymph node positivity was the only predictor of postoperative recurrence (odds ratio, 2.2; 95% confidence interval, 1.38-2.99; p = 0.007). Higher rates of lymphovascular invasion (p = 0.02), perineural invasion (p < 0.001), and R1 margin involvement (p < 0.001), as seen in one institution, did not result in poorer long-term survival in terms of the overall (p = 0.43), SPN-specific (p = 0.69), and recurrence-free survivals (p = 0.067). CONCLUSIONS: In contrast to previous findings that SPNs are prevalent in young women, a racial predilection for middle-aged Asian men and a delayed female peak incidence were noted. Parenchyma-preserving pancreatectomy may be an acceptable treatment. Non-radical surgery may be appropriate in patients with multiple comorbidities.

Is the Rationale of Anatomical Liver Resection for Hepatocellular Carcinoma Universally Adoptable? A Hypothesis-Driven Review.

Surgical resection is the first-line curative treatment modality for resectable hepatocellular carcinoma (HCC). Anatomical resection (AR), described as systematic removal of a liver segment confined by tumor-bearing portal tributaries, may improve survival by reducing the risk of tumor recurrence compared with non-AR. In this article, we propose the rationale for AR and its universal adoption by providing supporting evidence from the advanced understanding of a tumor microenvironment and accumulating clinical experiences of locoregional tumor ablation therapeutics. AR may be advantageous because it completely removes the en-bloc by interrupting tumor vascular supply and thus extirpates the spreading of tumor microthrombi, if they ever exist, within the supplying portal vein. However, HCC is a hypervascular tumor that can promote neoangiogenesis in the local tumor microenvironment, which in itself can break through the anatomical boundary within the liver and even retrieve nourishment from extrahepatic vessels, such as inferior phrenic or omental arteries. Additionally, increasing clinical evidence for locoregional tumor ablation therapies, such as radiofrequency ablation, predominantly performed as a non-anatomical approach, suggests comparable outcomes for surgical resection, particularly in small HCC and colorectal, hepatic metastases. Moreover, liver transplantation for HCC, which can be considered as AR of the whole liver followed by implantation of a new graft, is not universally free from post-transplant tumor recurrence. Overall, AR should not be considered the gold standard among all surgical resection methods. Surgical resection is fundamentally reliant on choosing the optimal margin width to achieve en-bloc tumor niche removal while balancing between oncological radicality and the preservation of postoperative liver function. The importance of this is to liberate surgical resilience in hepatocellular carcinoma. The overall success of HCC treatment is determined by the clearance of the theoretical niche. Developing biomolecular-guided navigation device/technologies may provide surgical guidance toward the total removal of microscopic tumor niche to achieve superior oncological outcomes.

Specific Bile Microorganisms Caused by Intra-Abdominal Abscess on Pancreaticoduodenectomy Patients: A Retrospective Cohort Study.

We retrospectively collected PD patients with a performance of bile culture between 2007 and 2019 in our institute. As to bile culture, we used a swab to do intraoperative bile cultures after transection of the CBD. IAA was defined as the documental bacteriological culture from either a turbid discharge from the intraoperatively placed drain in patients with a clinical picture consistent with infection or a postoperative fluid collection managed by CT-guided placement of drains. A total of 1244 PD patients were identified, and 539 (43.3%) subjects with bile sampling were included for analysis. Among these study patients, 433 (80.3%) developed bile contamination (positive bile culture). Bile contamination showed a significantly higher rate of IAA compared to non-bile contamination (17.1% vs. 0.9%, p < 0.001). The rate of co-shared microorganisms in both bile and abscess was 64.1%. On the multivariate analysis, age and specific bile microorganisms (Enterococcus species, Escherichia Coli, Streptococcus species, Citrobacter species, and Candida) are significantly associated with development of IAA. Specific bile microorganisms are the highly significant factors associated with development of IAA. The strategy to prevent bile spillage during PD should be considered to minimize afterward contamination of the abdominal cavity and prevent IAA.

Thoracoscopic Lobectomy Versus Sublobar Resection for pStage I Geriatric Non-Small Cell Lung Cancer.

OBJECTIVES: The choice of resection method for geriatric patients with early-stage non-small cell lung cancer (NSCLC) remains contentious. This study aimed to evaluate survival and perioperative outcomes after thoracoscopic lobectomy resection (LR) or sublobar resection (SR) in patients aged ≥75 years with pathologic stage (pStage) I NSCLC. MATERIALS AND METHODS: We retrospectively examined 258 consecutive patients aged ≥75 years with pStage I NSCLC who underwent thoracoscopic tumor resection at our institute from 2011 to 2018. Propensity score matching (PSM) analysis identified 60 patients in each group for comparison of survival-related parameters, including disease-free survival (DFS), lung cancer-specific overall survival (OS), and non-lung cancer-specific OS, using the Kaplan-Meier analysis. RESULTS: LR and SR were performed in 84 (32.6%) and 174 (67.4%) patients aged ≥75 years, respectively. The LR group had younger patients, better performance status, larger tumor sizes, and deeper tumor location than the SR group. Multivariate studies showed that the resection method was not a prognostic factor for OS. The two PSM-matched groups were not significantly different with respect to lung cancer-specific OS (p = 0.116), non-lung cancer-specific OS (p = 0.408), and DFS (p = 0.597). SR helped achieve better perioperative outcomes than LR, including fewer postoperative complications (10.0% vs. 28.3%, p = 0.011), shorter operative times (p < 0.001), decreased blood loss (p = 0.026), and shorter chest tube duration (p = 0.010) and hospital stays (p = 0.035). CONCLUSIONS: Thoracoscopic SR may provide similar oncological outcomes to LR, but may be a safer and more feasible surgical method for geriatric patients with pStage I NSCLC.

Stereotactic Breast Biopsy With Benign Results Does Not Negatively Affect Future Screening Adherence.

PURPOSE: To evaluate whether false-positive stereotactic vacuum-assisted breast biopsy (SVAB) affects subsequent mammographic screening adherence. MATERIALS AND METHODS: This Institutional Review Board-approved, HIPAA-compliant retrospective review of women with SVAB was performed between 2012 and 2014. Patient age, clinical history, biopsy pathology, and first postbiopsy screening mammogram were reviewed. Statistical analyses were performed using Fisher's exact, Mann-Whitney, and χ2 tests. RESULTS: There were 913 SVABs performed in 2012 to 2014 for imaging detected lesions; of these, malignant or high-risk lesions or biopsies resulting in a recommendation of surgical excision were excluded, leaving 395 SVABs yielding benign pathology in 395 women. Findings were matched with a control population consisting of 45,126 women who had a BI-RADS 1 or 2 screening mammogram and did not undergo breast biopsy. In all, 191 of 395 (48.4%) women with a biopsy with benign results and 22,668 of 45,126 (50.2%) women without biopsy returned for annual follow-up >9 months and ≤18 months after the index examination (P = .479). In addition, 57 of 395 (14.4%) women with a biopsy with benign results and 3,336 of 45,126 (7.4%) women without biopsy returned for annual follow-up >18 months after the index examination (P < .001). Older women, women with personal history of breast cancer, and women with postbiopsy complication after benign SVAB were more likely to return for screening (P = .026, P = .028, and P = .026, respectively). CONCLUSION: The findings in our study suggest that SVABs with benign results do not negatively impact screening mammography adherence. The previously described "harms" of false-positive mammography and biopsy may be exaggerated.

Significant symptoms alleviation and tumor volume reduction after combined simultaneously integrated inner-escalated boost and volumetric-modulated arc radiotherapy in a patient with unresectable bulky hepatocellular carcinoma: A care-compliant case report.

BACKGROUND: Clinically, elderly patients with unresectable bulky hepatocellular carcinoma (HCC) are difficult to manage, especially in those with co-infections of hepatitis B and C virus. Herein, we reported such a case treated with radiotherapy (RT) by using combined simultaneously integrated inner-escalated boost and volumetric-modulated arc radiotherapy (SIEB-VMAT). After RT, significant symptoms alleviation and durable tumor control were observed. CASE SUMMARY: At presentation, an 85-year-old male patient complained abdominal distention/pain, poor appetite, and swelling over bilateral lower limbs for 1 month. On physical examination, a jaundice pattern was noted. Laboratory studies showed impaired liver and renal function. Abdominal computed tomography (CT) revealed a 12.5-cm bulky tumor over the caudate lobe of the liver. Biopsy was done, and hepatocellular carcinoma (HCC) was reported histopathologically. As a result, AJCC stage IIIA (cT3aN0M0) and BCLC stage C were classified. Surgery, radiofrequency ablation (RFA), trans-catheter arterial chemoembolization (TACE), and sorafenib were not recommended because of his old age, central bulky tumor, and a bleeding tendency. Thus, RT with SIEB-VMAT technique was given alternatively. RT was delivered in 26 fractions, with dose gradience as follows: 39 Gy on the outer Plan Target Volume (PTV), 52 Gy in the middle PTV, and 57.2 Gy in the inner PTV. Unexpectedly, cyproheptadine (a newly recognized potential anti-HCC agent) was retrospectively found to be prescribed for alleviating skin itching and allergic rhinitis since the last 2 weeks of the RT course (2 mg by mouth Q12h for 24 months).After RT, significant symptoms alleviation and tumor volume reduction were observed for 32 months till multiple bone metastases. Before and after RT, a large tumor volume reduction rate of 88.7% was observed (from 608.4 c.c. to 68.7 c.c.). No severe treatment toxicity was noted during and after RT. The patient died due to aspiration pneumonia with septic shock at 4 months after bone metastases identified. CONCLUSIONS: SIEB-VMAT physically demonstrated double benefits of intratumor dose escalation and extra-tumor dose attenuation. Significant tumor regression and symptoms alleviation were observed in this elderly patient with unresectable bulky HCC. Further prospective randomized trials are encouraged to demarcate effective size of SIEB-VMAT with or without cyproheptadine.

Zeranol induces deleterious effects on the testes and the prostate gland of mature rats.

BACKGROUND: Endocrine disrupters have been shown to affect the male and female reproductive systems and to alter potential fertility. OBJECTIVES: This study was conducted to evaluate the effect of a continuous-release pellet containing 12 mg of zeranol for 30 days on the testes and the prostate gland of mature male rats. RESULTS: Zeranol treatment induced significant decrease of the testes and the prostate gland weights which were associated with a remarkable atrophy of the testicular seminiferous tubules and prominent regression of the glandular compartment of the prostate gland. However, zeranol treatment increased the thickness of the periductal layer of stromal cells of the prostate gland from a thin layer that express intense immunostaining of SM-actin and mild vimentin to a thicker layer of cells that exhibited intense immunostaining for both SM-actin and vimentin. CONCLUSION: These findings suggest that zeranol-induced changes to the prostate gland could result from either a direct effect of zeranol on the prostate gland or an indirect effect by interfering with testosterone production through disruption of testicular function.

(-)-Gossypol-enriched cottonseed oil inhibits proliferation and adipogenesis of human breast pre-adipocytes.

BACKGROUND: Breast cancer is the most commonly diagnosed cancer in women. Obesity is an important risk factor for developing breast cancer and is one of few risk factors that women can modify to prevent cancer. (-)-Gossypol-enriched cottonseed oil [(-)-GPCSO] contains 65% (-)-gossypol and 35% (+)-gossypol. Previous studies have demonstrated that both (-)-gossypol and (-)-GPCSO have potent anticancer activity against multiple types of cancer, including breast cancer. In addition, (-)-GPCSO reduced body weight gain and food intake in young female rats. However, the role of (-)-GPCSO on adipogenesis in human breast pre-adipocytes remains unclear. MATERIALS AND METHODS: Primary human breast pre-adipocytes were induced to differentiate in adipogenic medium in the presence of (-)-GPCSO. The proliferation of pre-adipocytes was determined with a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2-H-tetrazolium (MTS) assay. Lipid accumulation and glycerol 3-phosphate dehydrogenase (GPDH) activity were measured during adipocyte differentiation. mRNA expression of cyclin-D1, B-cell lymphoma-2 (BCL-2), Peroxisome Proliferator-Activated Receptor-γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα) and leptin was analyzed by real-time PCR. RESULTS: (-)-GPCSO inhibited proliferation of pre-adipocytes and down-regulated the expression of cyclin-D1 and BCL-2. (-)-GPCSO also significantly decreased adipogenesis, as determined by inhibition of GPDH activity, triglyceride content (TG), and down-regulation of the expression of PPARγ, C/EBPα and leptin. CONCLUSION: These findings suggest that (-)-GPCSO has the potential as a food supplement to inhibit adipogenesis, and therefore, reduce obesity.

Testosterone effect on the expression of genes that mediate testosterone metabolism and genes that mediate the effect of those metabolites on the prostate.

AIMS: The aim of this study was to investigate the effect of testosterone treatment on the proliferation index and the mRNA expression levels of 5α-reductase, CYP7B1, androgen receptor (AR), and estrogen receptor ß (ΕRß) in the canine prostate. MAIN METHODS: Immature dogs were treated with testosterone for one month, after which prostate gland growth was assessed by comparing the proliferation index in prostates from testosterone-treated dogs with that of untreated control dogs. The relative mRNA expression levels of the aforementioned genes in the prostate glands of testosterone-treated and untreated dogs were determined by real time PCR. KEY FINDINGS: Testosterone treatment induced a highly significant reduction in proliferation index in prostate gland. This inhibition of prostate gland growth was associated with differential mRNA expression of 5α-reductase, CYP7B1, AR, and ΕRß by the prostate gland of testosterone-treated dogs, as compared to that of untreated dogs. While the expression levels of 5α-reductase and CYP7B1 mRNA were significantly down-regulated by testosterone treatment, the expression level of ER-ß mRNA was highly up-regulated. In contrast, AR mRNA expression was not significantly altered. SIGNIFICANCE: Prostate gland proliferation appeared to be associated with the expression levels of genes that encode proteins that control intra-prostatic levels of testosterone metabolites and their respective receptors. Testosterone treatment may regulate gene expression in the prostate to generate a phenotype that suppresses growth-promoting signaling through AR and enhances anti-proliferative signaling through ERß. Therefore, targeting disturbances of this genetic machinery in benign prostate hyperplasia and prostate cancer is of a therapeutic potential.

Liposomes containing (-)-gossypol-enriched cottonseed oil suppress Bcl-2 and Bcl-xL expression in breast cancer cells.

PURPOSE: We have demonstrated that (-)-gossypol-enriched cottonseed oil [(-)-GPCSO] can down-regulate Bcl-2 expression in MCF-7 and primary cultured human breast cancer epithelial cells (PCHBCECs). However, this agent has not been evaluated in vivo due to its limited solubility. We aimed to develop liposomes containing (-)-GPCSO to suppress Bcl-2/Bcl-xL expression. METHODS: (-)-GPCSO liposomes were prepared and evaluated for effects on breast cancer cell viability, MDA-MB-231 xenograft tumor growth, cellular Bcl-2 and Bcl-xL mRNA levels, and chemosensitivity to paclitaxel. RESULTS: (-)-GPCSO liposomes prepared had excellent stability. Cytotoxicity of (-)-GPCSO liposomes was significantly reduced compared to (-)-GPCSO in culture medium. Bcl-2 and Bcl-xL mRNA expression was down-regulated by (-)-GPCSO in culture medium or (-)-GPCSO liposomes in MDA-MB-231 cells. In PCHBCECs, Bcl-2 and Bcl-xL expression were down-regulated by (-)-GPCSO liposomes. (-)-GPCSO in culture medium induced only a mild reduction in Bcl-xL. In the MDA-MB-231 xenograft tumor model, (-)-GPCSO liposomes exhibited tumor-suppressive activity and significantly reduced intratumoral Bcl-2 and Bcl-xL expression. Cytotoxicity of paclitaxel was increased by pretreatment with (-)-GPCSO liposomes in MDA-MB-231 and PCHBCECs. CONCLUSIONS: Findings suggest that (-)-GPCSO liposomes warrant continued investigation as a chemosensitizer for breast cancers exhibiting Bcl-2-/Bcl-xL-mediated drug resistance.

Zeranol down-regulates p53 expression in primary cultured human breast cancer epithelial cells through epigenetic modification.

Epidemiological studies have suggested that there are many risk factors associated with breast cancer. Silencing tumor suppressor genes through epigenetic alterations play critical roles in breast cancer initiation, promotion and progression. As a growth promoter, Zeranol (Z) has been approved by the FDA and is widely used to enhance the growth of beef cattle in the United States. However, the safety of Z use as a growth promoter is still under debate. In order to provide more evidence to clarify this critical health issue, the current study investigated the effect of Z on the proliferation of primary cultured human normal and cancerous breast epithelial cells (PCHNBECs and PCHBCECs, respectively) isolated from the same patient using MTS assay, RT-PCR and Western blot analysis. We also conducted an investigation regarding the mechanisms that might be involved. Our results show that Z is more potent to stimulate PCHBCEC growth than PCHNBEC growth. The stimulatory effects of Z on PCHBCECs and PCHBCECs may be mediated by its down-regulating expression of the tumor suppressor gene p53 at the mRNA and protein levels. Further investigation showed that the expression of DNA methylatransferase 1 mRNA and protein levels is up-regulated by treatment with Z in PCHBCECs as compared to PCHNBECs, which suggests a role of Z in epigenetic modification involved in the regulation of p53 gene expression in PCHBCECs. Our experimental results imply the potentially adverse health effect of Z in breast cancer development. Further study is continuing in our laboratory.

Zeranol induces cell proliferation and protein disulfide isomerase expression in mammary gland of ACI rat.

BACKGROUND: Zeranol is a non-steroidal anabolic growth promoter with potent estrogenic activity that is widely used as a growth promoter in the US beef industry. Consumption of beef derived from Zeranol-implanted cattle may be a risk factor for breast cancer. Protein disulfide isomerase (PDI) has been studied extensively as a key enzyme involving in the formation of the correct pattern of disulfide bonds in newly synthesized proteins. The relationship between PDI expression and cancer development has attracted interest of cancer researchers in recent years. MATERIALS AND METHODS: We implanted ACI rats with 12 mg Zeranol pellet and harvested the mammary tissues and tumor at day 110 after implantation and investigated the effect of Zeranol-implantation on cell proliferation by histological examination and proliferation in vitro. We also evaluated PDI mRNA expression in primary epithelial cells isolated from normal mammary glands and primary tumor cells from tumor specimens using real-time RT-PCR. To further confirm, we also evaluated the effect of Zeranol on PDI mRNA expression in primary epithelial cells isolated from normal mammary gland of ACI rats. RESULTS: We observed a palpable mammary tumor in one of three Zeranol-implanted ACI rats at day-110 post Zeranol-implantation. Zeranol-implantation significantly promoted the cell proliferation of primary mammary epithelial and stromal cells isolated from the mammary gland of normal ACI rats. PDI mRNA is over-expressed in primary tumor cells isolated from the tumor specimen and in Zeranol-treated primary cultured epithelial cells from the mammary gland of normal ACI rats. CONCLUSION: These findings suggest that up-regulated expression of PDI may play a critical role in mammary tumorigenesis and cell proliferation in response to Zeranol. Our findings implicate PDI as a biomarker for mammary tumorigenesis.

Serum derived from zeranol-implanted ACI rats promotes the growth of human breast cancer cells in vitro.

BACKGROUND: Zeranol (Z) is a non-steroidal anabolic growth promoter with potent estrogenic activity that is widely used as a growth promoter in the US beef industry. Consumption of beef derived from zeranol-implanted cattle may be a risk factor for breast cancer. MATERIALS AND METHODS: The effect of serum on the proliferation of human breast cancer MCF-7 cell line and primary cultured human breast epithelial cells (PCHBECs) was investigated. ACI rats were implanted with 12 mg zeranol pellet and the serum was harvested at day 110 after implantation. The effect of zeranol-serum on mRNA expression of cell cycle regulating gene (cyclin D1) and tumor suppressor genes (p53, and p21) was evaluated using real-time RT-PCR. RESULTS: The serum derived from ACI rats 110 days post-zeranol implantation significantly promoted the proliferation of MCF-7 cells and primary cultured human breast epithelial cells compared to control serum. Zeranol-serum up-regulated cyclin D1 and down-regulated p53 and p21 expression in PCHBECs compared with control serum. CONCLUSION: Bio-active zeranol metabolites contained in meat produced from cattle after zeranol implantation may be a risk factor for breast cancer.

Host-viral effects of chromatin assembly factor 1 interaction with HCMV IE2.

Chromatin assembly factor 1 (CAF1) consisting of p150, p60 and p48 is known to assemble histones onto newly synthesized DNA and thus maintain the chromatin structure. Here, we show that CAF1 expression was induced in human cytomegalovirus (HCMV)-infected cells, concomitantly with global chromatin decondensation. This apparent conflict was thought to result, in part, from CAF1 mislocalization to compartments of HCMV DNA synthesis through binding of its largest subunit p150 to viral immediate-early protein 2 (IE2). p150 interaction with p60 and IE2 facilitated HCMV DNA synthesis. The IE2Q548R mutation, previously reported to result in impaired HCMV growth with unknown mechanism, disrupted IE2/p150 and IE2/histones association in our study. Moreover, IE2 interaction with histones partly depends on p150, and the HCMV-induced chromatin decondensation was reduced in cells ectopically expressing the p150 mutant defective in IE2 binding. These results not only indicate that CAF1 was hijacked by IE2 to facilitate the replication of the HCMV genome, suggesting chromatin assembly plays an important role in herpesviral DNA synthesis, but also provide a model of the virus-induced chromatin instability through CAF1.

In vitro transformation of MCF-10A cells by sera harvested from heifers two months post-Zeranol implantation.

Among many risk factors of breast cancer, estrogens and non-estrogenic endocrine disruptors are considered to play critical roles in human breast carcinogenesis. Zeranol (Z) is a non-steroidal agent with potent estrogenic activity and has been widely used as an FDA approved beef growth promoter in the US. Recently, concerns have been raised about the potential adverse health risk by consumption of products containing biologically active Z and its metabolites. By utilizing cell proliferation assay, soft agar assay, quantitative real-time PCR and Western blotting analysis, we examined the potentially tumorigenic activity of bio-active Z containing sera harvested from heifers two months post Z-implantation and the underlying mechanisms. Our results showed that the growth of MCF-10A exposed to 0.2, 1 and 5% Z-containing serum (ZS) treatment for 3 weeks was 1.3, 1.75 and 1.8-fold faster compared to that of the control sera. After further investigation, we found that ZS increased cyclin D1 and decreased p53 expression at the mRNA and protein levels in MCF-10A compared to the controls. More importantly, treatment of 1% Z-containing sera for 21 days stimulated MCF-10A cells anchorage-independent colony formation in soft agar which illustrates its capability of inducing human normal breast epithelial cell neoplastic transformation. Our experimental results suggest that long-term exposure of low levels of Z and its metabolites contained in beef products might be a potential risk factor in human breast cancer initiation and development.