Induction of CTH expression in response to amino acid starvation confers resistance to anti-LAT1 therapy in MDA-MB-231 cells.

L type amino acid transporter 1 (LAT1) is an attractive molecular target for cancer therapy because of its overexpression in many cancer cells. JPH203, a selective LAT1 inhibitor, causes amino acid deprivation and suppresses cancer cell proliferation. However, several cancer cells showed resistance to amino acid deprivation. In this study, we aimed to elucidate the molecular mechanism of different sensitivity between 2 breast cancer cells to anti-LAT1 therapy. MDA-MB-231 cells were more resistant to growth suppression effect of JPH203 than T-47D cells (IC50 was 200 ± 12.5 µM for MDA-MB-231, and 5 ± 1.1 µM for T-47D cells; p < 0.05). Transcriptome and biochemical analysis were done in these cells in the presence/absence of JPH203. JPH203 induced intracellular amino acid deprivation stress in both cells, but it upregulated cystathionine γ lyase (CTH), an enzyme for synthesis of antioxidants, only in MDA-MB-231 cells. Moreover, siRNA-mediated CTH knockdown induced oxidative stress in response to JPH203 leading to decreased cell viability in MDA-MB-231 cells. These results suggest that activation of anti-oxidation pathways in response to amino acid deprivation confers resistance to anti-LAT1 therapy.

Expression of L-Type Amino Acid Transporter 1 is a Predictive Biomarker of Intravesical Recurrence in Patients with Non-Muscle Invasive Bladder Cancer.

PURPOSE: L-type amino acid transporter 1 (LAT1), a Na+-independent amino acid transporter, is highly expressed in various cancer types. We evaluated the prognostic value of LAT1 expression in non-muscle-invasive bladder cancer (NMIBC). PATIENTS AND METHODS: We retrospectively reviewed 119 consecutive patients who underwent initial transurethral resection of bladder tumor. Of these, 75 patients with NMIBC were included in this study. Patients were classified into two groups according to the proportion of LAT1-positive cells, as determined by immunohistochemistry. Associations between LAT1 expression and clinicopathological factors were analyzed. Cox multivariate analyses were performed to identify independent predictors of intravesical recurrence (IVR). The LAT1 integrated risk model was compared with the European Organization for Research and Treatment of Cancer (EORTC) risk model to evaluate the predictive ability for IVR based on the c-index. RESULTS: The median follow-up was 37 months. Twenty-eight patients (37.3%) had IVR. LAT1 expression was not correlated with any other clinicopathological factors. Patients with high LAT1 expression had a worse IVR-free survival than that of patients with low LAT1 expression (P = 0.038). Cox multivariate analyses indicated that tumor multiplicity and high LAT1 expression were independent predictors of IVR. The LAT1 integrated risk model had a significantly improved performance over the EORTC model for assessing recurrence risk (c-index: 0.695, improvement: 0.091, P = 0.001). When patients were stratified into three groups according to the score calculated by the LAT1 integrated risk model, the 2-year IVR-free survival rates were 93.3% in patients with 0 points, 66.9% for those with 2 points, and 37.5% for those with 4 points. CONCLUSION: High LAT1 expression was an independent predictor of IVR in patients with NMIBC. The LAT1 integrated risk model had good predictability for IVR.

Equine nonneoplastic abnormal ovary in a draft mare with high serum anti-Müllerian hormone: a case study.

We performed a standing hand-assisted laparoscopic ovariectomy in a draft mare that presented with high serum anti-Müllerian hormone (AMH) level and had an enlarged single cystic ovary. Histopathological examination revealed no tumor cell proliferation in the ovary, but the presence of a large ovarian cyst was confirmed. In the diagnosis of abnormal ovaries in mares, a comprehensive assessment should be performed, including the monitoring of ovarian morphology and biomarkers over time, to determine the disease prognosis and treatment plan. The case of this mare with a nonneoplastic abnormal ovary and increased serum AMH level was rare. We suggest that standing hand-assisted laparoscopic ovariectomy is useful for the removal of large ovaries in draft mares.

Prostate Cancer Cells in Different Androgen Receptor Status Employ Different Leucine Transporters.

BACKGROUND: Leucine stimulates cancer cell proliferation through the mTOR pathway, therefore, inhibiting leucine transporters may be a novel therapeutic target for cancer. L-type amino acid transporter (LAT) 1, a Na+ -independent amino acid transporter, is highly expressed in many tumor cells. However, leucine transporter(s) in different stages of prostate cancer, particularly in the stages of castration resistance with androgen receptor (AR) expression, is unclear. METHODS: LNCaP and DU145 and PC-3 cell lines were used as a model of androgen dependent, and metastatic prostate cancer. A new "LN-cr" cell line was established after culturing LNCaP cells for 6 months under androgen-free conditions, which is considered a model of castration resistant prostate cancer (CRPC) with androgen AR expression. The expression of leucine transporters was investigated with quantitative PCR and immunofluorescence. Uptake of 14 C Leucine was examined in the presence or absence of BCH (a pan-LAT inhibitor), JPH203 (an LAT1-specific inhibitor), or Na+ . Cell growth was assessed with MTT assay. siRNA studies were performed to evaluate the indispensability of y+ LAT2 on leucine uptake and cell viability in LN-cr. RESULTS: Cell viability showed a 90% decrease in the absence of leucine in all four cell lines. LNCaP cells principally expressed LAT3, and their leucine uptake was more than 90% Na+ -independent. BCH, but not JPH203, inhibited leucine uptake, and cell proliferation (IC50BCH :15 mM). DU145 and PC-3 cells predominantly expressed LAT1. Leucine uptake and cell growth were suppressed by BCH or JPH203 in a dose-dependent manner (IC50BCH : ∼20 mM, IC50JPH203 : ∼5 µM). In LN-cr cells, Na+ -dependent uptake of leucine was 3.8 pmol/mgprotein/min, while, Na+ -independent uptake was only 0.52 (P < 0.05). Leucine uptake of LN-cr was largely (∼85%) Na+ -dependent. y+ LAT2 expression was confirmed in LN-cr. Knockdown of y+ LAT2 lead to significant leucine uptake inhibition (40%) and cell growth inhibition (20%). CONCLUSIONS: New CRPC cell line with increased expression of y+ LAT2 as a leucine transporter was established in vitro. Anti-leucine transporter therapy could be an important option against prostate cancer. Prostate 77:222-233, 2017. © 2016 Wiley Periodicals, Inc.

Metformin enhances anti-tumor effect of L-type amino acid transporter 1 (LAT1) inhibitor.

BACKGROUND: In many cancer cells, L-type amino acid transporter 1 (LAT1) transports neutral amino acids with bulky side chain, which activate mammalian target of rapamycin (mTOR) to cause cell proliferation. An anti-diabetic drug, metformin, has been shown to activate AMP-activated protein kinase (AMPK), which leads to inhibition of mTOR. LAT1 inhibition in combination with metformin could result in more prominent suppression of mTOR activity. PURPOSE: Anti-proliferative effect of a newly developed LAT1 specific inhibitor JPH203 in combination with metformin is evaluated in 2 head and neck cancer cell lines, Ca9-22 and HEp-2 cells and in nude mice inoculated with Ca9-22 cells. RESULTS AND DISCUSSION: By MTT assay, 0.5 mM metformin inhibited proliferation of Ca9-22 cells to 70% of control. In the presence of 100 µM JPH203, proliferation of Ca9-22 cells was inhibited to 60% of control. By combining these 2 drugs, proliferation of Ca9-22 was significantly inhibited to 40% of control. However, this regimen was not very effective against HEp-2 cells. This combination also suppressed in vivo growth of Ca9-22 cells in a xenotransplant model. A combination of anti-LAT1 drug with metformin may be an effective anti-proliferative therapy for certain subsets of cancers.

Minimally invasive proximal interphalangeal joint arthrodesis using a locking compression plate and tissue engineering in horses: a pilot study.

This pilot study assessed the efficacy of 2 minimally invasive techniques for proximal interphalangeal (PIP) joint arthrodesis in horses. The PIP joints of both forelimbs (n = 6) were stabilized with locking compression plates (LCP) using a minimally invasive technique (LCP technique). Subsequently, for 1 randomly selected PIP joint of each horse, surgical drilling (SurD) was performed and tissue engineering (TE) was applied (LCP/SurD/TE technique). Minimally invasive PIP joint arthrodesis with LCP demonstrated low postoperative infection rates. Gross and histological evaluations revealed considerable destruction of the articular cartilage in the LCP/SurD/TE-treated joints. In contrast, almost no destruction of the cartilage was observed in the LCP-treated joints. Our results suggest that the LCP technique alone is not sufficient for PIP joint arthrodesis and that the LCP/SurD/TE technique may be useful for PIP joint arthrodesis in horses.

Arthrodèse de l'articulation interphalangienne proximale à effraction minimale à l'aide d'une plaque de fixation à compression et de l'ingénierie tissulaire chez les chevaux : une étude pilote. Cette étude pilote a évalué l'efficacité de 2 techniques à effraction minimale pour l'arthrodèse de l'articulation interphalangienne proximale (AIP) chez les chevaux. Les articulations AIP des deux membres antérieurs (n = 6) ont été stabilisées avec des plaques de fixation à compression (PFC) à l'aide d'une technique à effraction minimale (technique PFC). Subséquemment, pour une articulation AIP choisie au hasard pour chaque cheval, un fraisage chirurgical (FC) a été réalisé et une ingénierie tissulaire (IT) a été appliquée (technique PFC/FC/IT). Une arthrodèse de l'articulation AIP à effraction minimale avec PFC a démontré de faibles taux d'infection postopératoire. Des évaluations brutes et histologiques ont révélé une destruction considérable du cartilage articulaire dans les articulations traitées à l'aide de la technique PFC/FC/IT. Par contraste, pratiquement aucune destruction du cartilage n'a été observée dans les articulations traitées par PCF. Nos résultats suggèrent que la technique PFC seule n'est pas suffisante pour l'arthrodèse des articulations AIP et que la technique PFC/FC/IT peut être utile pour l'arthrodèse de l'articulation AIP chez les chevaux.(Traduit par Isabelle Vallières).

In vitro biomechanical comparison of a 5-hole 4.5 mm locking compression plate and 5-hole 4.5 mm dynamic compression plate for equine proximal interphalangeal joint arthrodesis.

OBJECTIVE: To compare the biomechanical properties of a 5-hole 4.5 mm narrow locking compression plate (LCP) and 5-hole 4.5 mm narrow dynamic compression plate (DCP) for equine proximal interphalangeal (PIP) joint arthrodesis. STUDY DESIGN: Experimental mechanical study. ANIMALS: Cadaveric adult equine forelimbs (n = 6 pair). METHODS: For each forelimb pair, 1 PIP joint was stabilized with LCP and the contralateral PIP joint with DCP. The 6 construct pairs were tested using a single-cycle, 3-point dorsopalmar bending system. PIP joints were evaluated with pre- and post-test radiography. RESULTS: The LCP technique had significantly greater yield load, failure load, and stiffness under single-cycle, 3-point dorsopalmar bending to failure than the DCP technique. There was no significant difference between the 2 constructs for displacement at yield and failure point. CONCLUSIONS: Biomechanically, the LCP technique provided significantly greater stability than the DCP technique under the test condition.