A case of nearly complete response in hepatocellular carcinoma with disseminated lung metastasis by combination therapy of nivolumab and ipilimumab after treatment failure of atezolizumab plus bevacizumab.

Recently, the efficacy of immuno-oncologic agents for advanced hepatocellular carcinoma (HCC) has been proven in several trials. In particular, atezolizumab with bevacizumab (AteBeva), as a first-line therapy for advanced HCC, has shown tremendous advances in the IMBrave150 study. However, second or third-line therapy after treatment failure with AteBeva has not been firmly established. Moreover, clinicians have continued their attempts at multidisciplinary treatment that includes other systemic therapy and radiotherapy (RT). Here, we report a case that showed a near complete response (CR) of lung metastasis to nivolumab with ipilimumab therapy after achieving a near CR of intrahepatic tumor using sorafenib and RT in a patient with advanced HCC who had experienced treatment failure of AteBeva.

Evaluation of Radiation Sensitivity Differences in Mouse Liver Tumor Organoids Using CRISPR/Cas9-Mediated Gene Mutation.

BACKGROUND: To assess the radiosensitivity of liver tumors harboring different genetic mutations, mouse liver tumors were generated in vivo through the hydrodynamic injection of clustered regularly interspaced short palindromic repeat/caspase 9 (CRISPR/Cas9) constructs encoding single-guide RNAs (sgRNAs) targeting Tp53, Pten, Nf1, Nf2, Tsc2, Cdkn2a, or Rb1. METHODS: The plasmid vectors were delivered to the liver of adult C57BL/6 mice via hydrodynamic tail vein injection. The vectors were injected into 10 mice in each group. Organoids were generated from mouse liver tumors. The radiation response of the organoids was assessed using an ATP cell viability assay. RESULTS: The mean survival period of mice injected with vectors targeting Nf2 (4.8 months) was lower than that of other mice. Hematoxylin and eosin staining, immunohistochemical (IHC) staining, and target sequencing analyses revealed that mouse liver tumors harbored the expected mutations. Tumor organoids were established from mouse liver tumors. Histological evaluation revealed marked morphological similarities between the mouse liver tumors and the generated tumor organoids. Moreover, IHC staining indicated that the parental tumor protein expression pattern was maintained in the organoids. The results of the ATP cell viability assay revealed that the tumor organoids with mutated Nf2 were more resistant to high-dose radiation than those with other gene mutations. CONCLUSIONS: This study developed a radiation response assessment system for mouse tumors with mutant target genes using CRISPR/Cas9 and organoids. The Tp53 and Pten double mutation in combination with the Nf2 mutation increased the radiation resistance of tumors. The system used in this study can aid in elucidating the mechanism underlying differential intrinsic radiation sensitivity of individual tumors.

Melittin regulates iron homeostasis and mediates macrophage polarization in rats with lumbar spinal stenosis.

Lumbar spinal stenosis (LSS) is defined as spinal canal narrowing, resulting in the compression of the nerves traversing the lower back into the leg. Inflammation is the most common cause of LSS. Elevated iron stores are often associated with chronic inflammation resulting in nerve damage-induced pain. Macrophage polarization to either the M1 (inflammatory) or M2 (anti-inflammatory) type is essential for regulating host defenses and promoting tissue repair. However, the precise role of macrophage polarization in iron release or retention in LSS pathophysiology remains elusive. Melittin, a component of bee venom, modulates iron metabolism-related macrophage polarization and is beneficial in LSS. We treated primary peritoneal macrophages with melittin and assessed macrophage polarization by immunofluorescence staining. Melittin (100 and 250 µg/kg) effects on iron deposition-induced macrophage polarization were also evaluated using immunochemistry, real-time PCR, and flow cytometry in an LSS rat model. Locomotor function was assessed using the Basso-Beattie-Bresnahan (BBB) locomotor rating scale, ladder scoring, and von Frey test for up to 3 weeks. Melittin induced M2 polarization of iron-insulted primary macrophages in vitro and increased the proportion of M2 macrophages in the damaged spinal cord in vivo. Moreover, melittin attenuated iron overload-induced M1 polarization by regulating iron metabolism-related genes in rats with LSS. In conclusion, melittin improves locomotor recovery and stimulates axonal growth following LSS. Additionally, it promotes functional recovery in LSS rat models by regulating macrophage iron metabolism, thereby activating M2 macrophages, suggesting its potential application in LSS treatment.

Harpagophytum procumbens Inhibits Iron Overload-Induced Oxidative Stress through Activation of Nrf2 Signaling in a Rat Model of Lumbar Spinal Stenosis.

Lumbar spinal stenosis (LSS) is a common degenerative spinal condition in older individuals that causes impaired walking and other disabilities due to severe lower back and leg pain. Ligamentum flavum hypertrophy is a major LSS cause that may result from oxidative stress caused by degenerative cascades, including imbalanced iron homeostasis that leads to excessive reactive oxygen species production. We investigated the effects of Harpagophytum procumbens (HP) on iron-induced oxidative stress associated with LSS pathophysiology. Primary spinal cord neuron cultures were incubated in FeSO4-containing medium, followed by addition of 50, 100, or 200 µg/mL HP. Cell viability was assessed by CCK-8 and live/dead cell assays and by propidium iodide-live imaging. In an in vivo rat model of LSS, HP were administered at 100, 200, and 400 mg/kg, and disease progression was monitored for up to 3 weeks. We investigated the in vitro and in vivo effects of HP on iron-induced neurotoxicity by immunochemistry, real-time PCR, and flow cytometry. HP exerted neuroprotective effects and enhanced neurite outgrowths of iron-injured rat primary spinal cord neurons in vitro. HP treatment significantly reduced necrotic cell death and improved cells' antioxidative capacity via the NRF2 signaling pathway in iron-treated neurons. At 1 week after HP administration in LSS rats, the inflammatory response and oxidative stress markers were substantially reduced through regulation of excess iron accumulation. Iron that accumulated in the spinal cord underneath the implanted silicone was also regulated by HP administration via NRF2 signaling pathway activation. HP-treated LSS rats showed gradually reduced mechanical allodynia and amelioration of impaired behavior for 3 weeks. We demonstrated that HP administration can maintain iron homeostasis within neurons via activation of NRF2 signaling and can consequently facilitate functional recovery by regulating iron-induced oxidative stress. This fundamentally new strategy holds promise for LSS treatment.

Correlation Between Average Segment Width and Gamma Passing Rate as a Function of MLC Position Error in Volumetric Modulated Arc Therapy.

OBJECTIVE: This study analyzed the correlation between the average segment width (ASW) and gamma passing rate according to the multi-leaf collimator (MLC) position error. METHOD: To evaluate the changes in the gamma passing rate according to the MLC position error, 21 volumetric modulated arc therapy (VMAT) plans were generated using pelvic lymph node metastatic prostate cancer patient's data which is sensitive to MLC position errors as they involve several long, narrow, irregular fields. The ASW for each VMAT plan was calculated using our own code developed using Visual Basic for Applications (VBA). The gamma passing rate of the VMAT plan according to the MLC position error was evaluated using ArcCHECK (Sun Nuclear, Melbourne, FL, USA) while inducing symmetric MLC position errors in 0.25 mm intervals from -1 mm to +1 mm in the infinity medical linear accelerator (Elekta AB, Stockholm, Sweden). Finally, we examined the correlation between the change in the passing rate (γgradient) due to the MLC position error and the ASW in VMAT through linear regression analysis using the least squares method. RESULTS: The ASW and γgradient were found to have a linear correlation according to the MLC position error, and the coefficient of determination was 0.88. For a 1 mm position error of MLC in VMAT, the gamma passing rate improved by approximately 11.9% as the ASW increased by 10 mm. CONCLUSION: These results are expected to be employed as guidelines to minimize the dose uncertainty due to MLC position error in VMAT.

Apamin Enhances Neurite Outgrowth and Regeneration after Laceration Injury in Cortical Neurons.

Apamin is a minor component of bee venom and is a polypeptide with 18 amino acid residues. Although apamin is considered a neurotoxic compound that blocks the potassium channel, its neuroprotective effects on neurons have been recently reported. However, there is little information about the underlying mechanism and very little is known regarding the toxicological characterization of other compounds in bee venom. Here, cultured mature cortical neurons were treated with bee venom components, including apamin, phospholipase A2, and the main component, melittin. Melittin and phospholipase A2 from bee venom caused a neurotoxic effect in dose-dependent manner, but apamin did not induce neurotoxicity in mature cortical neurons in doses of up to 10 µg/mL. Next, 1 and 10 µg/mL of apamin were applied to cultivate mature cortical neurons. Apamin accelerated neurite outgrowth and axon regeneration after laceration injury. Furthermore, apamin induced the upregulation of brain-derived neurotrophic factor and neurotrophin nerve growth factor, as well as regeneration-associated gene expression in mature cortical neurons. Due to its neurotherapeutic effects, apamin may be a promising candidate for the treatment of a wide range of neurological diseases.

Evaluation of monoxide film-based dosimeters for surface dose detection in electron therapy.

Generally, electron therapy is applied to tumors on or close to the skin surface. However, this causes a variety of skin-related side effects. To alleviate the risk of these side effects, clinical treatment uses skin dosimeters to verify the therapeutic dose. However, dosimeters suffer from poor accuracy, because their attachment sites are approximated with the help of naked eyes. Therefore, a dosimeter based on a flexible material that can adjust to the contours of the human body is required. In this study, the reproducibility, linearity, dose-rate dependence, and percentage depth ionization (PDI) of PbO and HgO film-based dosimeters are evaluated to explore their potential as large-scale flexible dosimeters. The results demonstrate that both dosimeters deliver impressive reproducibility (within 1.5%) and linearity (≥ 0.9990). The relative standard deviations of the dose-rate dependence of the PbO and HgO dosimeters were 0.94% and 1.16% at 6 MeV, respectively, and 1.08% and 1.25% at 9 MeV, respectively, with the PbO dosimeter outperforming the 1.1% of existing diodes. The PDI analysis of the PbO and HgO dosimeters returned values of 0.014 cm (-0.074 cm) and 0.051 cm (-0.016 cm), respectively at 6 MeV (9 MeV) compared to the thimble chamber and R50. Therefore, the maximum error of each dosimeter is within the allowable range of 0.1 cm. In short, the analysis reveals that the PbO dosimeter delivers a superior performance relative to its HgO counterpart and has strong potential for use as a surface dosimeter. Thus, flexible monoxide materials have the necessary qualities to be used for dosimeters that meet the requisite quality assurance standards and can satisfy a variety of radiation-related applications as flexible functional materials.

Neurotherapeutic Effect of Inula britannica var. Chinensis against H2O2-Induced Oxidative Stress and Mitochondrial Dysfunction in Cortical Neurons.

Inula britannica var. chinensis (IBC) has been used as a traditional medicinal herb to treat inflammatory diseases. Although its anti-inflammatory and anti-oxidative effects have been reported, whether IBC exerts neuroprotective effects and the related mechanisms in cortical neurons remain unknown. In this study, we investigated the effects of different concentrations of IBC extract (5, 10, and 20 µg/mL) on cortical neurons using a hydrogen peroxide (H2O2)-induced injury model. Our results demonstrate that IBC can effectively enhance neuronal viability under in vitro-modeled reaction oxygen species (ROS)-generating conditions by inhibiting mitochondrial ROS production and increasing adenosine triphosphate level in H2O2-treated neurons. Additionally, we confirmed that neuronal death was attenuated by improving the mitochondrial membrane potential status and regulating the expression of cytochrome c, a protein related to cell death. Furthermore, IBC increased the expression of brain-derived neurotrophic factor and nerve growth factor. Furthermore, IBC inhibited the loss and induced the production of synaptophysin, a major synaptic vesicle protein. This study is the first to demonstrate that IBC exerts its neuroprotective effect by reducing mitochondria-associated oxidative stress and improving mitochondrial dysfunction.

Bee Venom Melittin Protects against Cisplatin-Induced Acute Kidney Injury in Mice via the Regulation of M2 Macrophage Activation.

Inflammation is an essential biological response that eliminates pathogenic bacteria and repairs tissue after injury. Acute kidney injury (AKI) is associated with systemic and intrarenal inflammation as the inflammatory process decreases renal function and promotes progression to advanced chronic kidney disease. Macrophages are key mediators of the inflammatory response; their activation influences the immune system and may have various effects. Classically activated type I macrophages (M1) produce a variety of pro-inflammatory cytokines at the lesion site. However, anti-inflammatory type II macrophages (M2) are alternatively activated upon exposure to anti-inflammatory cytokines and are associated with wound healing and tissue repair following AKI. Here, we used melittin from bee venom to enhance the polarization of M2 macrophages and promote renal recovery after AKI. Melittin was administered to mice intraperitoneally for 5 days at various concentrations (10, 50, and 100 µg/kg); serum creatinine and blood urea nitrogen (BUN) levels were analyzed 72 h after cisplatin administration to confirm renal dysfunction. Melittin inhibited the cisplatin-induced increase in creatinine and BUN, an indicator of renal dysfunction. The expression of M1 markers (CD16/32) decreased significantly, whereas that of M2 markers (CD206, Arg1nase I) increased after melittin administration. Consistently, tubular necrosis was substantially reduced in melittin-treated mice. Thus, melittin alleviates cisplatin-induced AKI by regulating M2 macrophage expression.

Which Is Better for Liver SBRT: Dosimetric Comparison Between DCAT and VMAT for Liver Tumors.

Stereotactic body radiotherapy (SBRT) is currently well-adopted as a curative treatment for primary and metastatic liver tumors. Among SBRT methods, dynamic conformal arc therapy (DCAT) and volumetric-modulated arc therapy (VMAT) are the most preferred methods. In this study, we report a comparison study measuring the dose distribution and delivery efficiency differences between DCAT and VMAT for liver SBRT. All patients who were treated with SBRT for primary or metastatic liver tumors with a curative aim between January 2016 and December 2017 at DIRAMS were enrolled in the study. For all patients, SBRT plans were designed using the Monte Carlo (MC) algorithm in Monaco treatment planning system (version 5.1). The planning goals were set according to the RTOG 0813, RTOG 0915, and RTOG 1112 protocols. A plan comparison was made on the metrics of dose volume histogram, planning and delivery efficiency, monitor unit (MU), and dosimetric indices. PTV coverage was evaluated using the following: Dmean, D95%, D98%, D2%, D50%, Dmax, V95%, heterogeneity index (HI), and conformality index (CI). For DCAT and VMAT, respectively, the Dmean was 5942.8 ± 409.3 cGy and 5890.6 ± 438.8 cGy, D50% was 5968.8 ± 413.1 cGy and 5954.3 ± 405.2 cGy, and CI was 1.05 ± 0.05 and 1.03 ± 0.04. The D98% and V95% were 5580.0 ± 465.3 cGy and 20.4 ± 12.0 mL for DCAT, and 5596.0 ± 478.7 cGy and 20.5 ± 12.0 mL for VMAT, respectively. For normal liver, V40, V30, V20, V17, V5, Dmean, Dmax were evaluated for comparison. The V30, V20, and V10 were significantly higher in DCAT; other parameters of normal livers showed no statistically significant differences. For evaluation of intermediate dose spillage, D2cm(%) and R50% of DCAT and VMAT were 45.8 ± 7.9 and 5.6 ± 0.9 and 45.1 ± 6.7 and 5.5 ± 1.2, respectively. Planning and delivery efficiency were evaluated using MU, Calculation time, and Delivery time. DCAT had shorter Calculation time and Delivery time with smaller MU. MU was smaller in DCAT and the average difference was 300.1 MU. For liver SBRT, DCAT is an effective alternative to VMAT plans that could meet the planning goals proposed by the RTOG SBRT protocol and increases plan and delivery effectiveness, while also ignoring the interplay effect.

Predictive impact of circulating microRNA-193a-5p on early relapse after autologous stem cell transplantation in patients with multiple myeloma.

We explored prognostic roles of circulating microRNAs (miRs) in multiple myeloma (MM) treated with autologous stem cell transplantation (ASCT) following induction chemotherapy. In part I of the study (n = 40), we identified a decreasing dynamics of circulating miR-193a-5p expression from diagnosis to pre-ASCT. In patients who experienced early relapse within one year post ASCT (n = 9) these patterns were distinctive compared to those without early relapse in a 1:2 matched cohort (n = 18). In part II (n = 90), multivariate analyses showed that the International Staging System score and miR-193a-5p expression before ASCT were independent prognostic factors. Conclusively, expression of circulating miR-193a-5p before ASCT could be a prognostic biomarker for transplant-eligible MM.

Direct electrophoretic microRNA preparation from clinical samples using nanofilter membrane.

A method to directly collect negatively charged nucleic acids, such as DNA and RNA, in the biosamples simply by applying an electric field in between the sample and collection buffer separated by the nanofilter membrane is proposed. The nanofilter membrane was made of low-stress silicon nitride with a thickness of 100 nm, and multiple pores were perforated in a highly arranged pattern using nanoimprint technology with a pore size of 200 nm and a pore density of 7.22 × 108/cm2. The electrophoretic transport of hsa-mir-93-5p across the membrane was confirmed in pure microRNA (miRNA) mimic solution using quantitative reverse transcription-polymerase chain reactions (qRT-PCR). Consistency of the collected miRNA quantity, stability of the system during the experiment, and yield and purity of the prepared sample were discussed in detail to validate the effectiveness of the electrical protocol. Finally, in order to check the applicability of this method to clinical samples, liquid biopsy process was demonstrated by evaluating the miRNA levels in sera of hepatocellular carcinoma patients and healthy controls. This efficient system proposed a simple, physical idea in preparation of nucleic acid from biosamples, and demonstrated its compatibility to biological downstream applications such as qRT-PCR as the conventional nucleic acid extraction protocols.

Treatment time to the end of thoracic radiotherapy has more predictive power for survival than radiation dose intensity in patients with limited-stage small-cell lung cancer receiving concurrent chemoradiation of more than 45 Gy.

The optimal protocol for thoracic radiotherapy (TRT) in combination with chemotherapy in patients with limited-stage small-cell lung cancer (LS-SCLC) remains elusive. The present study aimed to evaluate radiation parameters in association with survival outcomes. A total of 101 patients with LS-SCLC who completed TRT at ≥45 Gy and concurrent chemotherapy were retrospectively reviewed. The median dose and duration of TRT were 50 Gy and 38 days, respectively. The median duration from the start of either therapy to the end of TRT (SER) was 60 days. The median survival for all patients was 26.9 months. The 3-year local control (LC), progression-free survival (PFS) and overall survival (OS) rates were 52.0, 29.5 and 37.6%, respectively, and the 5-year LC, PFS and OS rates were 50.1, 28.3 and 26.7%, respectively. Univariate analysis revealed that patient age, tumor stage, timing and dose of TRT, SER, prophylactic cranial irradiation (PCI), and tumor response were significantly associated with treatment outcomes. Multivariate analysis revealed that stage was the only significant prognostic factor for LC (P=0.011), PFS (P<0.001) and OS (P<0.001). Tumor response (P=0.014), PCI (P=0.007) and SER (P=0.005) were significant predictors of OS. OS was improved in patients who achieved complete response, and their SER was ≤70 days (P<0.001). Short treatment duration (SER ≤70 days) was a significant predictor of OS in patients with LS-SCLC who completed planned TRT at ≥45 Gy with concurrent chemoradiotherapy.

CRTC2 suppresses BMP2-induced osteoblastic differentiation via Smurf1 expression in MC3T3-E1 cells.

AIMS: CREB (cAMP response element-binding protein)-regulated transcription coactivator (CRTC2) has been reported to act as a coactivator of CREB during gluconeogenesis. The role of CRTC2 in osteoblastic differentiation has not yet been elucidated. The aim of this study is to identify the mechanism of CRTC2 in osteoblast differentiation. MAIN METHODS: The mRNA expression was determined by RT-PCR and qPCR. Protein levels were measured using Western blot assay. Alkaline phosphatase (ALP) staining was performed to evaluate ALP activity. Alizarin red S (ARS) staining was performed to measure extracellular mineralization. Transcriptional activity was detected using a luciferase assay. KEY FINDINGS: In the present study, TNF-α was found to stimulate CRTC2 expression. However, TNF-α did not increase the gene expression of osteoblast differentiation markers and inhibited BMP2-induced osteoblastic differentiation. Overexpression of CRTC2 decreased the expression of osteogenic genes, ALP activity and extracellular matrix mineralization. Knockdown of CRTC2 restored BMP2-induced osteogenic gene expression and ALP activity. CRTC2 increased Smurf1 mRNA expression, Smurf 1 promoter activity, and protein level. Furthermore, Smurf 1 decreased Smad 1/5/9 protein levels. These results suggest that CRTC2 decreased BMP2-induced osteoblastic differentiation via Smurf 1 expression. SIGNIFICANCE: Our results indicate that CRTC2 regulates the expression of Smurf1 in osteoblast differentiation.

Analysis of survival outcomes based on molecular subtypes in breast cancer brain metastases: A single institutional cohort.

PURPOSE: To evaluate the survival outcomes based on molecular subtypes of breast cancer in patients with brain metastasis. MATERIALS AND METHODS: We retrospectively reviewed 106 breast cancer patients treated for brain metastases, from January 2005 to May 2016. Patients were divided into four groups based on the tumor molecular subtype: luminal A (Estrogen Receptor [ER]/Progesterone Receptor [PR] positive, human epithelial growth factor receptor-2 [HER2] negative), luminal B (ER/PR positive, HER2 Positive), HER2 (HER2 positive and ER/PR negative), and Triple negative (TNBC). RESULTS: The median follow-up time for surviving patients was 22 months (range: 11.2-51.1 months). The median survival of all patients was 14 months, with a 1-year overall survival (OS) rate of 57.5% and a 2-year OS rate of 32.1%. Thirty patients (28.3%) had a solitary brain metastasis while 62 (58.5%) patients had multiple metastases. A significant difference was observed in the survival rates of the two groups. Based on the Karnofsky performance score, the performance status of the patients at the time of brain metastasis was also found to affect survival. Patients with different molecular subtypes had different survival rates; the luminal A group showed the highest median survival (luminal A: 23.1, luminal B: 15.0, HER2: 12.5 and TNBC: 6.4 months, respectively), which was statistically significant. CONCLUSION: In breast cancer patients with brain metastasis, survival rates were different based on the molecular subtype of the tumor, despite various local and systemic treatments. Appropriate and tailored treatment approaches should, therefore, be considered for the different molecular subtypes.

Correlation of biologically effective dose and the tumor control in Stage I (<5 cm) non-small cell lung cancer with stereotactic ablative radiotherapy: a single institutional cohort study.

BACKGROUNDS: Stereotactic ablative radiotherapy (SABR) is one of the newly developed innovative radiotherapy and of which optimal dose prescription needs to be standardized. We aimed to investigate the dose-response relationship for patients with SABR. METHODS: Fifty-three patients with Stage I non-small cell lung cancer patients, who underwent SABR between November 2006 and January 2015, were evaluated retrospectively. Thirteen patients (24.5%), who refused the surgery were included and 40 patients (75.5%) were medically inoperable at diagnosis. The median age was 74 years. The median SABR dose was 50 Gy in 3-8 fractions and the median biologically effective dose (BED;α/ß = 10) was 105.6 Gy (range: 60-160.53 Gy). RESULTS: The median follow-up was 37.1 months. The 1 and 3 year local control rates were 91.7% and 85.1%. The 3 year overall and progression-free survival rate were 63.3% and 47.5%, respectively, and freedom from progression was 62.2%. Local control rate and 3-year overall survival according to tumor size was 100% and 79.4% in T1 tumors in a while 61.8% and 45% in T2a tumors. The 3-year local and regional control by BED10 was 79.4% and 69.4% in ≤100 Gy vs. 89.1% and 100% in >100 Gy (P = 0.526, 0.004). Dyspnea more than Grade 3 was reported in six (11.3%) patients and Grade 1 chest pain was shown in five (9.4%) patients. CONCLUSIONS: The excellent regional control was conferred with a prescription of more than BED10 of 100 Gy, which also might be needed to achieve better local tumor control in T2a patients with tolerable lung function.

Costunolide increases osteoblast differentiation via ATF4-dependent HO-1 expression in C3H10T1/2 cells.

AIMS: Costunolide is a sesquiterpene lactones used in many herbal medicines, with well-established anti-inflammatory and anti-oxidant functions modulating endoplasmic reticulum (ER) stress pathways, and which promotes the expression of anti-oxidant genes. The aim of this study is to investigate whether costunolide is involved in osteoblast differentiation and, determine the mechanisms of differentiation in mesenchymal stem cells. MAIN METHODS: The cytotoxicity of costunolide was identified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The mRNA and protein expression levels of osteogenic genes were determined by RT-PCR and Western blot analysis. Alkaline phosphate (ALP) staining and Alizarin red S (ARS) staining were performed to evaluate ALP activity and matrix mineralization. Transcriptional activity was detected using a luciferase reporter assay. KEY FINDINGS: In this study, we determined that costunolide increased the expression of distal-less homeobox 5 (Dlx5), runt-related transcription factor 2 (Runx2), ALP, and osteocalcin (OC) in C3H10T 1/2 cells. Furthermore, costunolide increased ALP activity and matrix mineralization. Interestingly, costunolide increased ER stress by Bip, activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP). However, it did not exert effects on expression of activating transcription factor 6 (ATF6). ATF4 activation has a protective role in oxidative stress, and its transcription induces anti-oxidant genes in cells. Heme oxygenase-1 (HO-1) is a major anti-oxidant enzyme, and is regulated by ATF4. We showed that costunolide treatment increased HO-1 expression. Furthermore, the HO-1 inhibitor, Sn(IV) Protoporphyrin IX dichloride (SnPP) was blocked costunolide-induced Runx2 expression. SIGNIFICANCE: Our results revealed that costunolide-induced osteoblast differentiation is regulated by ATF4-dependent HO-1 expression.

Prognostic Significance of Inner Quadrant Involvement in Breast Cancer Treated with Neoadjuvant Chemotherapy.

PURPOSE: In the present study, we aimed to evaluate the initial tumor location as a prognostic factor in breast cancer patients treated with neoadjuvant chemotherapy (NAC). METHODS: Between March 2002 and January 2007, a total of 179 patients with stage II/III breast cancer underwent NAC followed by breast surgery. Using physical and radiologic findings, patients were grouped by their initial tumor location into inner/both quadrant (upper/lower inner quadrant involvement +/- multicentric tumor involving outer quadrant; n=97) and outer quadrant (n=82) tumor groups. All patients received neoadjuvant docetaxel/doxorubicin chemotherapy. One hundred two patients underwent modified radical mastectomy and 77 patients underwent breast-conserving surgery. Adjuvant radiotherapy (RT) and hormonal therapy were administered after surgery when indicated. While 156 patients underwent postoperative RT, 23 did not. The median follow-up duration was 61.1 (12-106) months. RESULTS: The 5-year disease-free survival (DFS) and overall survival rates of all patients were 74.8% and 89.9%, respectively. Patients with inner/both quadrant tumors had lower 5-year DFS than those with outer quadrant tumors (67.7% vs. 83.4%, respectively; hazard ratio [HR]=1.941, p=0.034). A nodal ratio >25% was also an independent adverse prognostic factor for DFS (HR=3.276; p<0.001). There was no significant difference in DFS (p=0.592) after RT on the internal mammary node (IMN). Treatment failed in 44 out of 179 patients (24.6%), of which 27 patients had inner/both quadrant tumors. Twenty-one out of 27 patients had distant failures. CONCLUSION: Among breast cancer patients treated with NAC, those with inner/both quadrant tumors had lower DFS than those with outer quadrant tumors. More aggressive neoadjuvant and/or adjuvant chemotherapy with IMN RT is required for improved disease control and long-term survival.

Outcome analysis of salvage radiotherapy for occult cervical cancer found after simple hysterectomy.

OBJECTIVE: The objective was to analyze the outcomes of the patients, who received salvage radiotherapy for incidentally discovered cervical cancer following simple hysterectomy, and to identify the influence of intracavitary radiotherapy on treatment outcomes. METHODS: Data from 117 patients with occult cervical cancer who underwent simple hysterectomy followed by salvage radiotherapy from September 1979 to November 2010 were collected. All the patients received external beam radiotherapy with (n = 45) or without (n = 72) intracavitary radiotherapy. Local control, disease-free survival, overall survival and treatment-related toxicity were investigated. RESULTS: The median follow-up time was 75 months. The 5- and 10-year local control/disease-free survival/overall survival rates were 93/87/87% and 90/84/83%, respectively. Among 98 patients who had no residual disease and negative resection margin on surgical specimens, 32 (33%) received intracavitary radiotherapy and 66 (67%) did not. There were no differences in patient and tumor characteristics between patients treated with and without intracavitary radiotherapy. The 5-year local control rate for the non-intracavitary radiotherapy group was 93 versus 94% for the intracavitary radiotherapy group (P = 0.564); the disease-free survival rate was 88 versus 94% (P = 0.894); the overall survival rate was 95 versus 85% (P = 0.106), respectively. Among all patients, there were 5% of Grade 3 or higher late toxicities. CONCLUSIONS: Patients with occult invasive cervical cancer discovered following simple hysterectomy could be treated safely and effectively with salvage radiotherapy. For patients with no residual disease and negative resection margin, intracavitary radiotherapy could be omitted.

Radial displacement of clinical target volume in node negative head and neck cancer.

PURPOSE: To evaluate the radial displacement of clinical target volume in the patients with node negative head and neck (H&N) cancer and to quantify the relative positional changes compared to that of normal healthy volunteers. MATERIALS AND METHODS: Three node-negative H&N cancer patients and five healthy volunteers were enrolled in this study. For setup accuracy, neck thermoplastic masks and laser alignment were used in each of the acquired computed tomography (CT) images. Both groups had total three sequential CT images in every two weeks. The lymph node (LN) level of the neck was delineated based on the Radiation Therapy Oncology Group (RTOG) consensus guideline by one physician. We use the second cervical vertebra body as a reference point to match each CT image set. Each of the sequential CT images and delineated neck LN levels were fused with the primary image, then maximal radial displacement was measured at 1.5 cm intervals from skull base (SB) to caudal margin of LN level V, and the volume differences at each node level were quantified. RESULTS: The mean radial displacements were 2.26 (±1.03) mm in the control group and 3.05 (±1.97) in the H&N cancer patients. There was a statistically significant difference between the groups in terms of the mean radial displacement (p = 0.03). In addition, the mean radial displacement increased with the distance from SB. As for the mean volume differences, there was no statistical significance between the two groups. CONCLUSION: This study suggests that a more generous radial margin should be applied to the lower part of the neck LN for better clinical target coverage and dose delivery.