Fei-Ting Hsu

Sorafenib increases efficacy of vorinostat against human hepatocellular carcinoma through transduction inhibition of vorinostat-induced ERK/NF-κB signaling

Sorafenib is effective for patients with advanced hepatocellular carcinoma (HCC) and particularly for those who are unsuitable to receive life-prolonging transarterial chemo-embolization. The survival benefit of sorafenib, however, is unsatisfactory. Vorinostat also known as suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase (HDAC) inhibitor with anti-HCC efficacy in preclinical studies. SAHA induces nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) activity in vitro, which may lead to cancer cell progression and jeopardize cytotoxic effect of SAHA in HCC. The goal of this study was to investigate whether sorafenib enhances SAHA cytotoxicity against HCC through inhibition of SAHA-induced NF-κB activity. The human HCC cell line Huh7 transfected with dual reporter genes, luciferase (luc) and thymidine kinase (tk) with NF-κB response elements, was co-transfected with red fluorescent protein (rfp) gene for non-invasive molecular imaging to assess NF-κB activity and living cells simultaneously. Cell viability assay, DNA fragmentation, western blotting, electrophoretic mobility shift assay (EMSA) and multiple modalities of molecular imaging were used to assess the combination efficacy and mechanism of sorafenib and SAHA. The administration of high-dose SAHA (10 µM) with long treatment time (48 h) in vitro, and 25 mg/kg/day by gavage in HCC-bearing nude mice to induce NF-κB activity were performed. Sorafenib inhibited SAHA-induced NF-κB activity and the expression of NF-κB-regulated effector proteins while it increased the efficacy of SAHA against HCC both in vitro and in vivo. The mechanism of sorafenib to enhance SAHA efficacy on HCC is through the suppression of ERK/NF-κB pathway, which induces extrinsic and intrinsic apoptosis. Combination of sorafenib and SAHA may have the potential as new strategy against HCC.

Decrease in Breast Density in the Contralateral Normal Breast of Patients Receiving Neoadjuvant Chemotherapy: MR Imaging Evaluation

PURPOSE To investigate the change of breast density with quantitative magnetic resonance (MR) imaging in the contralateral normal breast of patients receiving neoadjuvant chemotherapy. MATERIALS AND METHODS This study was approved by the institutional review board and was HIPAA compliant. Informed consent was obtained. Fifty-four patients with breast cancer (mean age, 47 years; age range, 30-74 years) treated with NAC protocol and enrolled in a breast MR imaging research study were studied. The density in the contralateral normal breast was analyzed by using an MR imaging-based segmentation method. The effect of chemotherapy on the change of density following the doxorubicin and cyclophosphamide (AC) and the AC and taxane regimen was evaluated. The dependence on age was investigated by using a multivariate regression model. RESULTS In patients who underwent both AC and taxane follow-up, the mean percentage of change from the individuals baseline density was -10% (95% confidence interval: -12.8%, -7.2%) after AC and -12.7% (95% confidence interval: -16%, -9.4%) after AC and taxane. In patients who underwent both follow-up studies after one to two and four cycles of AC, the mean percentage of change was -9.4% (95% confidence interval: -13.5%, -5.3%) after one to two cycles of AC and -14.7% (95% confidence interval: -20.6%, -8.7%) after four cycles of AC. The percentage reduction of density was significantly dependent on age. Patients younger than 40 years had a greater reduction after chemotherapy than patients older than 55 years (P = .01). CONCLUSION By using three-dimensional MR imaging, patients receiving chemotherapy showed reduction of breast density, and the effects were significant after initial treatment with one to two cycles of the AC regimen.

Using NF-κB as a molecular target for theranostics in radiation oncology research.

Resistance of cancer cells to chemotherapy and/or radiotherapy is a major challenge to current anticancer treatment. The NF-κB signaling pathway plays an important role in tumor development and progression, and results in unsatisfactory treatment outcome. Inhibition of the NF-κB signaling cascade may sensitize the resistant cancer cells to chemotherapy and/or radiotherapy. Here, the correlation of NF-κB molecules with carcinogenesis and tumor progression, along with its significance in clinical practice, is reviewed. The potential clinical application of NF-κB and its associated molecules as diagnostic and therapeutic targets is also discussed.

Curcumin synergistically enhances the radiosensitivity of human oral squamous cell carcinoma via suppression of radiation-induced NF-κB activity

The anticancer effect of curcumin has been widely reported. However, whether curcumin can enhance the radiosensitivity of human oral squamous cell carcinoma (OSCC) remains to be elucidated. The aim of the present study was to evaluate the efficacy of curcumin combined with radiation against OSCC. SAS cells were transfected with the luciferase gene (luc) and named SAS/luc. NF-κB/DNA binding activity, the surviving fraction and NF-κB-regulated effector protein expression were determined by electrophoretic mobility shift assay, clonogenic survival assay and western blotting, respectively. The therapeutic efficacy was evaluated in SAS/luc tumor-bearing mice by caliper measurement and bioluminescence imaging. Curcumin enhanced SAS/luc radiosensitivity through the inhibition of radiation-induced NF-κB activity and expression of effector proteins both in vitro and in vivo. With 4 Gy or greater radiation doses, synergistic effects of curcumin were observed. The combination group (curcumin plus radiation) had significantly better tumor control compared with that of curcumin or radiation alone. No significant body weight change of mice was found throughout the entire study. In conclusion, curcumin is a radiosensitizer against OSCC with negligible toxicity.

Enhancement of adoptive T cell transfer with single low dose pretreatment of doxorubicin or paclitaxel in mice

Ex vivo expansion of CD8+ T-cells has been a hindrance for the success of adoptive T cell transfer in clinic. Currently, preconditioning with chemotherapy is used to modulate the patient immunity before ACT, however, the tumor microenvironment beneficial for transferring T cells may also be damaged. Here preconditioning with single low dose of doxorubicin or paclitaxel combined with fewer CD8+ T-cells was investigated to verify whether the same therapeutic efficacy of ACT could be achieved. An E.G7/OT1 animal model that involved adoptive transfer of OVA-specific CD8+ T-cells transduced with a granzyme B promoter-driven firefly luciferase and tomato fluorescent fusion reporter gene was used to evaluate this strategy. The result showed that CD8+ T-cells were activated and sustained longer in mice pretreated with one low-dose Dox or Tax. Enhanced therapeutic efficacy was found in Dox or Tax combined with 2×106 CD8+ T-cells and achieved the same level of tumor growth inhibition as that of 5×106 CD8+ T-cells group. Notably, reduced numbers of Tregs and myeloid derived suppressor cells were shown in combination groups. By contrast, the number of tumor-infiltrating cytotoxic T lymphocytes and IL-12 were increased. The NF-κB activity and immunosuppressive factors such as TGF-β, IDO, CCL2, VEGF, CCL22, COX-2 and IL-10 were suppressed. This study demonstrates that preconditioning with single low dose Dox or Tax and combined with two fifth of the original CD8+ T-cells could improve the tumor microenvironment via suppression of NF-κB and its related immunosuppressors, and activate more CD8+ T-cells which also stay longer.

Does breast density show difference in patients with estrogen receptor-positive and estrogen receptor-negative breast cancer measured on MRI?

Mammographic density is a known risk factor for breast cancer. Studies have shown that women with increased mammographic density have higher risk of developing breast cancer compared with women with lower mammographic density [1, 2]. Both endogenous and exogenous estrogen may influence mammographic density. Mammographic density decreases after menopause when ovarian function declines. Hormonal replacement therapy, with combination of estrogen and progesterone, increases mammographic density [2], while tamoxifen, which has antiestrogenic effect, decreases mammographic density [3]. Mammographic density therefore can be regarded as a marker of the effect of estrogen on the breast tissue. To what extent mammographic density is a predictor for both hormone receptor-positive and hormone receptor-negative tumors is still unclear. Postmenopausal hormonal replacement therapy is associated with an increased risk of developing an estrogen receptor (ER)-positive tumor [4]. In symptomatic breast cancers, a significant positive association was found between ER and progesterone receptor (PgR) status of the tumors and percent density [5]. If an increased risk of ER-positive breast cancer is associated with breast density, it may be possible to find a higher density in patients who develop ER-positive cancer than ER-negative cancer. In this study, we investigated the breast density using three-dimensional (3D) magnetic resonance imaging (MRI)-based method [6] in two groups of patients, with ER-positive and ER-negative cancer. The evaluation of breast density based on mammogram may not accurately analyze the density due to projection imaging with the tissue-overlapping problem. MRI, however, provides strong soft tissue contrast distinguishing between fibroglandular and fatty tissues and a 3D view of breast tissues without compression. In a retrospective review of breast cancer patients who received breast MRI at our institution from 2004 to 2006, 80 pathologically proven patients with unilateral invasive ductal carcinoma and with complete information of ER and PgR status were included and studied (12 Asian women and 68 white women; 45 ER/PgR positive and 35 ER/PgR negative). These 80 subjects came from a sub-cohort of women in our another study focusing on the comparison of breast density between invasive ductal carcinoma and ductal carcinoma in situ. The breast density was analyzed from the contralateral normal breast of each subject, assuming symmetric bilateral breast density. It was noted that density measurements of the two breasts in women are highly correlated [7]. This study was approved by the institutional review board and was Health Insurance Portability and Accountability compliant. All patients gave written informed consent for receiving the MRI study. The breast MRI was carried out in a 1.5-T magnetic resonance (MR) with a 4-channel phased-array bilateral breast coil (Philips Medical Systems, Cleveland, OH). The imaging protocol consisted of pre-contrast T1-weighted imaging and bilateral dynamic contrast-enhanced imaging using a 3D spoiled gradient recalled radiofrequency-spoiled (RF)-fast acquisition at steady rate (FAST) pulse sequence. Thirty-two axial images covering both breasts were acquired. Noncontrast 3D SPGR (RF-FAST) T1-weighted images without fat suppression (repetition time = 8.1 ms, echo time = 4.0 ms, flip angle = 20°, matrix size = 256 × 128, field of view = 38 cm, slice thickness = 3–4 mm) were used to calculate the breast density. In this study, since tumor volume will affect the measurement of the breast density, the contralateral breast not harboring tumor was selected for density measurement. Breast density measurement was carried out based on our recently developed MR method [6]. With this method, the breast and the fibroglandular tissue were segmented using computer-assisted algorithms. After the breast was segmented out, the total breast volume was calculated. The adaptive Fuzzy C-Means was applied for segmentation of the fibroglandular tissue from the surrounding fatty tissue. After completing the segmentation from all 2D imaging slices, the volume of fibroglandular tissue was calculated, and the percent density was obtained by normalizing to the total breast volume. The mean age was 54 years in the ER-positive group and 47 years in the ER-negative group (significantly younger, P < 0.005). In the ER-positive group, 20 patients were ≥55 years old and 25 were <55 years old. Overall, the measured breast density did not show significant difference between the ER-positive and ER-negative patient groups (9.9% ± 7.2% for the ER-positive group versus 12.6% ± 8.9% for the ER-negative group, P = 0.14). Figure 1 shows the scattered plot between the percent breast density and the age for the two groups of patients. It was noted that there were no obvious differences among patients with different cancer types. However, the age dependence was clearly noted, higher density with younger age. A logistic model was applied to analyze the difference in density, controlling for age, and the results show no significant difference between ER-positive and ER-negative cancers. Figure 2 shows two patients with ER-positive and ER-negative breast cancer, who had similar breast density in the contralateral normal breast. Figure 1. Scatter plot of percent breast density versus age in patients with ER-positive (ER+) and ER-negative (ER−) breast cancer. A clear age dependence is noted, but not between the two cancer types. ER, estrogen receptor. Figure 2. (A) A 41-year-old Hispanic woman with ER-positive cancer; (B) a 50-year-old Hispanic woman with ER-negative cancer. ER-positive cancer is a mass lesion, and ER-negative cancer is a nonmass lesion, indicated by arrows. The segmented fibroglandular density ... Although mammographic density is a risk factor for breast cancer, it is not clear whether mammographic density can predict subtypes of breast cancer defined by expression of the their hormonal and human epidermal growth factor receptor 2 receptors. To clarify if increased mammographic density is related to ER status of breast cancer, studies from literature regarding if increased mammographic density is related to ER status of breast cancer showed conflicting results [4, 8, 9]. A study using Breast Imaging Reporting and Data System categories 1–4 for classifying mammographic density has found that increasing density was significantly correlated with negative ER status [8]; another study with same approach, however, showed no correlation of increased density with the ER status of the tumor [9]. Using quantitative mammographic percent density, increased mammographic density was associated with both ER-positive and ER-negative breast cancer, and both luminal type and triple-negative cancer, indicating breast density did not predict tumor type [4, 10]. This study was the first one to use 3D MR-based method for quantification of breast density between different cancer types. Our results did not show significant differences in breast density between ER-positive and ER-negative patients, indicating that the link between breast density and breast cancer may be due to factors other than or in addition to estrogen exposure [4].

Curcumin Sensitizes Hepatocellular Carcinoma Cells to Radiation via Suppression of Radiation-Induced NF-κB Activity

The effects and possible underlying mechanism of curcumin combined with radiation in human hepatocellular carcinoma (HCC) cells in vitro were evaluated. The effects of curcumin, radiation, and combination of both on cell viability, apoptosis, NF-κB activation, and expressions of NF-κB downstream effector proteins were investigated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), NF-κB reporter gene, mitochondrial membrane potential (MMP), electrophoretic mobility shift (EMSA), and Western blot assays in Huh7-NF-κB-luc2, Hep3B, and HepG2 cells. Effect of I kappa B alpha mutant (IκBαM) vector, a specific inhibitor of NF-κB activation, on radiation-induced loss of MMP was also evaluated. Results show that curcumin not only significantly enhances radiation-induced cytotoxicity and depletion of MMP but inhibits radiation-induced NF-κB activity and expressions of NF-κB downstream proteins in HCC cells. IκBαM vector also shows similar effects. In conclusion, we suggest that curcumin augments anticancer effects of radiation via the suppression of NF-κB activation.

Erlotinib-Conjugated Iron Oxide Nanoparticles as a Smart Cancer-Targeted Theranostic Probe for MRI

We designed and synthesized novel theranostic nanoparticles that showed the considerable potential for clinical use in targeted therapy, and non-invasive real-time monitoring of tumors by MRI. Our nanoparticles were ultra-small with superparamagnetic iron oxide cores, conjugated to erlotinib (FeDC-E NPs). Such smart targeted nanoparticles have the preference to release the drug intracellularly rather than into the bloodstream, and specifically recognize and kill cancer cells that overexpress EGFR while being non-toxic to EGFR-negative cells. MRI, transmission electron microscopy and Prussian blue staining results indicated that cellular uptake and intracellular accumulation of FeDC-E NPs in the EGFR overexpressing cells was significantly higher than those of the non-erlotinib-conjugated nanoparticles. FeDC-E NPs inhibited the EGFR–ERK–NF-κB signaling pathways, and subsequently suppressed the migration and invasion capabilities of the highly invasive and migrative CL1-5-F4 cancer cells. In vivo tumor xenograft experiments using BALB/c nude mice showed that FeDC-E NPs could effectively inhibit the growth of tumors. T2-weighted MRI images of the mice showed significant decrease in the normalized signal within the tumor post-treatment with FeDC-E NPs compared to the non-targeted control iron oxide nanoparticles. This is the first study to use erlotinib as a small-molecule targeting agent for nanoparticles.

Synergistic Effect of Sorafenib and Radiation on Human Oral Carcinoma in vivo

Oral squamous cell carcinoma often causes bone invasion resulting in poor prognosis and affects the quality of life for patients. Herein, we combined radiation with sorafenib, to evaluate the combination effect on tumor progression and bone erosion in an in situ human OSCC-bearing mouse model. Treatment procedure were arranged as following groups: (a) normal (no tumor); (b) control (with tumor); (c) sorafenib (10 mg/kg/day); (d) radiation (single dose of 6 Gy); (e) pretreatment (sorafenib treatment for 3 days prior to radiation), and (f) concurrent treatment (sorafenib and radiation on the same day). The inhibition of tumor growth and expression level of p65 of NF-κB in tumor tissues were the most significant in the pretreatment group. EMSA and Western blot showed that DNA/NF-κB activity and the expressions of NF-κB-associated proteins were down-regulated. Notably, little to no damage in mandibles and zygomas of mice treated with combination of sorafenib and radiation was found by micro-CT imaging. In conclusion, sorafenib combined with radiation suppresses radiation-induced NF-κB activity and its downstream proteins, which contribute to radioresistance and tumorigenesis. Additionally, bone destruction is also diminished, suggesting that combination treatment could be a potential strategy against human OSCC.

Regorafenib induces extrinsic and intrinsic apoptosis through inhibition of ERK/NF-κB activation in hepatocellular carcinoma cells

The aim of the present study was to investigate the role of NF-κB inactivation in regorafenib-induced apoptosis in human hepatocellular carcinoma SK-HEP-1 cells. SK-HEP-1 cells were treated with different concentrations of the NF-κB inhibitor 4-N-[2-(4-phenoxyphenyl)ethyl]quinazoline-4,6-diamine (QNZ) or regorafenib for different periods. The effects of QNZ and regorafenib on cell viability, expression of NF-κB-modulated anti-apoptotic proteins and apoptotic pathways were analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, western blotting, DNA gel electrophoresis, flow cytometry and NF-κB reporter gene assay. Inhibitors of various kinases including AKT, c-Jun N-terminal kinase (JNK), P38 and extracellular signal-regulated kinase (ERK) were used to evaluate the mechanism of regorafenib-induced NF-κB inactivation. The results demonstrated that both QNZ and regorafenib significantly inhibited the expression of anti-apoptotic proteins and triggered extrinsic and intrinsic apoptosis. We also demonstrated that regorafenib inhibited NF-κB activation through ERK dephosphorylation. Taken all together, our findings indicate that regorafenib triggers extrinsic and intrinsic apoptosis through suppression of ERK/NF-κB activation in SK-HEP-1 cells.