Expression of Semaphorin 3F and Its Receptors in Epithelial Ovarian Cancer, Fallopian Tubes, and Secondary Müllerian Tissues

While semaphorins and their receptors appear to play a role in tumor carcinogenesis, little is known about the role of semaphorin 3F (S3F) in epithelial ovarian cancer (EOC) development. Therefore, we sought to determine the clinical relationship between S3F and its receptors, neuropilin-2 (NP-2) and neuropilin-1 (NP-1) with EOC progression. We analyzed the immunohistological expression of S3F, NP-2, and NP-1 in clinical specimens of normal ovaries (N), benign cystadenomas (Cy), well-differentiated adenocarcinomas (WD), poorly-differentiated adenocarcinomas (PD), inclusion cysts (IC), paraovarian cysts (PC), and fallopian tubes (FT). Tissue sections were evaluated for staining intensity and percentage of immunoreactive epithelia. We found that expression of S3F and NP-2 decreased while NP-1 expression increased with EOC progression. Interestingly, we also found elevated expression of S3F, NP-2, and NP-1 in epithelia of ICs, PCs, and FT. Our findings indicate that loss or deregulation of semaphorin signaling may play an important role in EOC development

A High-Throughput Screen Indicates Gemcitabine and JAK Inhibitors May be Useful for Treating Pediatric AML

Improvement in survival has been achieved for children and adolescents with AML but is largely attributed to enhanced supportive care as opposed to the development of better treatment regimens. High risk subtypes continue to have poor outcomes with event free survival rates \u3c 40% despite the use of high intensity chemotherapy in combination with hematopoietic stem cell transplant. Here we combine high-throughput screening, intracellular accumulation assays, and in vivo efficacy studies to identify therapeutic strategies for pediatric AML. We report therapeutics not currently used to treat AML, gemcitabine and cabazitaxel, have broad anti-leukemic activity across subtypes and are more effective relative to the AML standard of care, cytarabine, both in vitro and in vivo. JAK inhibitors are selective for acute megakaryoblastic leukemia and significantly prolong survival in multiple preclinical models. Our approach provides advances in the development of treatment strategies for pediatric AML

Expression of Semaphorin 3F and Its Receptors in Epithelial Ovarian Cancer, Fallopian Tubes, and Secondary Müllerian Tissues

While semaphorins and their receptors appear to play a role in tumor carcinogenesis, little is known about the role of semaphorin 3F (S3F) in epithelial ovarian cancer (EOC) development. Therefore, we sought to determine the clinical relationship between S3F and its receptors, neuropilin-2 (NP-2) and neuropilin-1 (NP-1) with EOC progression. We analyzed the immunohistological expression of S3F, NP-2, and NP-1 in clinical specimens of normal ovaries (N), benign cystadenomas (Cy), well-differentiated adenocarcinomas (WD), poorly-differentiated adenocarcinomas (PD), inclusion cysts (IC), paraovarian cysts (PC), and fallopian tubes (FT). Tissue sections were evaluated for staining intensity and percentage of immunoreactive epithelia. We found that expression of S3F and NP-2 decreased while NP-1 expression increased with EOC progression. Interestingly, we also found elevated expression of S3F, NP-2, and NP-1 in epithelia of ICs, PCs, and FT. Our findings indicate that loss or deregulation of semaphorin signaling may play an important role in EOC development

Panobinostat Enhances Cytarabine and Daunorubicin Sensitivities in AML Cells through Suppressing the Expression of BRCA1, CHK1, and Rad51

<div><p>Acute myeloid leukemia (AML) remains a challenging disease to treat and urgently requires new therapies to improve its treatment outcome. In this study, we investigated the molecular mechanisms underlying the cooperative antileukemic activities of panobinostat and cytarabine or daunorubicin (DNR) in AML cell lines and diagnostic blast samples <i>in vitro</i> and <i>in vivo</i>. Panobinostat suppressed expression of BRCA1, CHK1, and RAD51 in AML cells in a dose-dependent manner. Further, panobinostat significantly increased cytarabine- or DNR-induced DNA double-strand breaks and apoptosis, and abrogated S and/or G2/M cell cycle checkpoints. Analogous results were obtained by shRNA knockdown of BRCA1, CHK1, or RAD51. Cotreatment of NOD-SCID-IL2Rγ^null mice bearing AML xenografts with panobinostat and cytarabine significantly increased survival compared to either cytarabine or panobinostat treatment alone. Additional studies revealed that panobinostat suppressed the expression of <i>BRCA1, CHK1,</i> and <i>RAD51</i> through downregulation of E2F1 transcription factor. Our results establish a novel mechanism underlying the cooperative antileukemic activities of these drug combinations in which panobinostat suppresses expression of <i>BRCA1, CHK1,</i> and <i>RAD51</i> to enhance cytarabine and daunorubicin sensitivities in AML cells.</p></div

Panobinostat suppresses <i>BRCA1</i>, <i>CHK1</i>, and <i>RAD51</i> protein and transcript expression and induces apoptosis in AML cell lines.

<p>THP-1 cells were treated with variable concentrations of panobinostat for 48 h. Whole cell lysates were subjected to Western blotting (<b>Panels A&D</b>). Total RNAs were isolated and mRNA levels were determined by Real-time RT-PCR (<b>Panel B</b>). Apoptotic events were determined by annexin V/PI staining and flow cytometry analysis (<b>Panel C</b>). Protein and mRNA levels for <i>BRCA1</i>, <i>CHK1</i>, and <i>RAD51</i> genes were determined by Western blotting (<b>Panel E</b>) and Real-time PCR (<b>Panel F</b>), respectively, in THP-1 cells treated with 10 nM panobinostat for up to 48 h. CTS, OCI-AML3 or U937 AML cells were treated with variable concentrations of panobinostat for 48 h. Whole cell lysates were subjected to Western blotting to measure protein levels for BRCA1, CHK1, and RAD51 in the cells (<b>Panel G</b>). The levels of apoptosis induced by panobinostat were determined by flow cytometry analysis with annexin V/PI staining (<b>Panel H</b>). Transcript levels for <i>BRCA1</i>, <i>CHK1</i>, and <i>RAD51</i> genes were determined by Real-time RT-PCR (<b>Panels I–K</b>).</p

Antileukemic activity of panobinostat alone, cytarabine alone, and panobinostat plus cytarabine in a U937 xenograft model.

<p>NOD-SCID-IL2Rγ^null (NSG) mice were injected with luciferase-labeled U937 cells and treated 3 days later with panobinostat (5 mg/kg once daily for 3 weeks), ara-C (6.25 mg/kg once daily for 4 weeks), or combination (panobinostat 5 mg/kg once daily for 3 weeks plus ara-C 6.25 mg/kg once daily for 4 weeks). Serial bioluminescence images of representative mice receiving panobinostat alone (n = 8), ara-C alone (n = 10), or panobinostat plus ara-C (n = 8) (<b>Panel A</b>). When bioluminescence reached 1.5×10⁷ p/s/cm²/sr, day 17 after injection of U937 cells, mice received panobinostat (5 mg/kg once daily×2). Four hours after the second dose mice were sacrificed and Bone Marrows were harvested. Pellets were lysed and subjected to Western Blot (<b>Panel B</b>). Tumor progression monitored by quantitative biophotonic imaging analysis of control and treatment groups (<b>Panel C</b>). A plot of overall survival probability, estimated with the Kaplan–Meier method (<b>Panel D</b>).</p

The roles of BRCA1, CHK1, and RAD51 in cytarabine- or DNR-induced DNA DSBs and apoptosis in THP-1 cells.

<p>THP-1 cells were infected with BRCA1, CHK1, RAD51, or non-target control (NTC) shRNA lentivirus overnight, washed and then treated with 4 µM cytarabine or 25 nM DNR for 48 h. shRNA knockdown of BRCA1, CHK1, or RAD51, and induction of γH2AX by cytarabine or DNR were determined by Western blotting (<b>Panels A–C</b>). The lane headings indicate the treatment conditions ‘Control’ ‘Ara-C’ or ‘DNR’ and the+or – indicate the shRNA-treated cells from which the sample was derived. Apoptotic events in the cells were determined by annexin V/PI staining and flow cytometry analyses (<b>Panel D</b>). THP-1 cells were infected with BRCA1, CHK1, or RAD51 shRNA lentivirus overnight. The cells were washed three times with complete medium and cultured in virus-free complete medium for up to 72 h. The cells were then treated with 50 µM cytarabine or 2 µM DNR for 3 h and the drugs were washed out, and the cells were cultured in drug-free complete medium for up to 8 h. DNA damage was assessed by COMET assay. Representative images at the 8 h time point are shown (<b>Panel E</b>). The median percent DNA in the tail from at least three replicate gels are shown plus or minus the standard error of the mean (Panel F). *indicates p<0.05.</p