BPR0C261, An Analogous of Microtubule Disrupting Agent D-24851 Enhances the Radiosensitivity of Human Non-Small Cell Lung Cancer Cells via p53-Dependent and p53-Independent Pathways.

(1) Destabilization of microtubule dynamics is a primary strategy to inhibit fast growing tumor cells. The low cytotoxic derivative of microtubule inhibitor D-24851, named BPR0C261 exhibits antitumor activity via oral administration. In this study, we investigated if BPR0C261 could modulate the radiation response of human non-small cell lung cancer (NSCLC) cells with or without p53 expression. (2) Different doses of BPR0C261 was used to treat human NSCLC A549 (p53+/+) cells and H1299 (p53-/-) cells. The cytotoxicity, radiosensitivity, cell cycle distribution, DNA damage, and protein expression were evaluated using an MTT assay, a colony formation assay, flow cytometry, a comet assay, and an immunoblotting analysis, respectively. (3) BPR0C261 showed a dose-dependent cytotoxicity on A549 cells and H1299 cells with IC50 at 0.38 µM and 0.86 µM, respectively. BPR0C261 also induced maximum G2/M phase arrest and apoptosis in both cell lines after 24 h of treatment with a dose-dependent manner. The colony formation analysis demonstrated that a combination of low concentration of BPR0C261 and X-rays caused a synergistic radiosensitizing effect on NSCLC cells. Additionally, we found that a low concentration of BPR0C261 was sufficient to induce DNA damage in these cells, and it increased the level of DNA damage induced by a fractionation radiation dose (2 Gy) of conventional radiotherapy. Furthermore, the p53 protein level of A549 cell line was upregulated by BPR0C261. On the other hand, the expression of PTEN tumor suppressor was found to be upregulated in H1299 cells but not in A549 cells under the same treatment. Although radiation could not induce PTEN in H1299 cells, a combination of low concentration of BPR0C261 and radiation could reverse this situation. (4) BPR0C261 exhibits specific anticancer effects on NSCLC cells by the enhancement of DNA damage and radiosensitivity with p53-dependent and p53-independent/PTEN-dependent manners. The combination of radiation and BPR0C261 may provide an important strategy for the improvement of radiotherapeutic treatment.

Ten-years outcome analysis in patients with clinically localized prostate cancer treated by radical prostatectomy or external beam radiation therapy.

Purpose: Since there was no consensus on treatment options for localized prostate cancer, we performed a retrospective study to compare the long-term survival benefit of radiotherapy (RT) versus laparoscopic radical prostatectomy (LRP) in Taiwan. Methods: 218 patients with clinically localized prostate cancer treated between 2008 and 2017 (64 with LRP and 154 with RT) were enrolled in this study. The outcomes of RT and LRP were assessed after patients were stratified according to Gleason score, stage, and risk group. Crude survival, prostate cancer-specific survival, and metastasis-free survival were evaluated using the log-rank test. Results: The 5-year crude survival rate was 93.3% in the LRP group and 59.3% in the RT group. A significant survival benefit was found in the LRP group compared with the RT group (p = 0.004). Furthermore, significant differences were found in disease-specific survival (93.3% vs. 64.7%, p = 0.022) and metastasis-free survival (48% vs. 40.2%, p = 0.045) between the LRP and RT groups. Conclusions: Men with localized prostate cancer treated initially with LRP had a lower risk of prostate cancer-specific death and metastases compared with those treated with RT.

Involvement of c-Myc in low dose radiation-induced senescence enhanced migration and invasion of unirradiated cancer cells.

Ionizing radiation is known to cause cell apoptosis at high dose range, but little is known about the cellular response to low dose radiation. In this study, we found that conditioned medium harvested from WI-38 lung fibroblasts and H1299 lung adenocarcinoma cells exposed to 0.1Gy to 1Gy could enhance the migration and invasion of unirradiated H1299 cells in both 2D and 3D culturing circumstances. Low dose radiation did not induce apoptosis, but induced senescence in irradiated cells. We next examined the expression of immediately early genes including c-Myc and K-Ras. Although both genes could be up-regulated by low dose radiation, induction of c-Myc was more specific to low dose range (0.5Gy) at transcriptional and translational levels. Knockdown of c-Myc by shRNA could repress the senescence induced by low dose radiation. The conditioned medium of irradiated cells induced migration of unirradiated cells was also repressed by knockdown of c-Myc. The c-Myc inhibitor 10058-F4 could suppress low dose radiation induced cell senescence, and the conditioned medium harvested from irradiated cells pretreated with 10058-F4 also lost the ability to enhance the migration of unirradiated cells. The cytokine array analysis revealed that immunosuppressive monocyte chemoattractant protein-1 increased by low dose radiation could be repressed by 10058-F4. We also showed that 10058-F4 could suppress low dose radiation induced tumor progression in a xenograft tumor model. Taken together, current data suggest that -Myc is involved in low dose radiation induced cell senescence and potent bystander effect to increase the motility of unirradiated cells.

Involvement of 8-O-acetylharpagide for Ajuga taiwanensis mediated suppression of senescent phenotypes in human dermal fibroblasts.

Herbal medicines are attractive agents for human care. In this study, we found that the alcohol extract of Ajuga taiwanensis (ATE) screened from a chemical bank exhibited potent capacity for suppressing senescence associated biomarkers, including SA-ß-gal and up-regulated p53 in old human dermal fibroblasts (HDFs) without induction of significant cytotoxicity up to 100 µg/ml. Concomitantly, cells re-entered the cell cycle by reducing G1 phase arrest and increasing cell growth rate. The ATE was further partitioned to obtain the sub-fractions of n-butanol (BuOH), ethyl acetate (EA) and water. The BuOH and water sub-fractions exhibited less effects on prohibition of cell growth than the EA sub-fraction. All of these sub-fractions exhibited the ability on suppressing SA-ß-gal and p53 of old HDFs as low as 5-10 µg/ml. Under the activity guided fractionation and isolation, a major active constituent named AT-1 was isolated. The AT-1 was further identified as 8-O-acetylharpagide by structural analysis, and it could suppress SA-ß-gal and p53 of old HDFs below 10 µM. In addition, the intracellular reactive oxygen species (ROS) levels of old HDFs were suppressed by ATE, the sub-fractions of BuOH and water, and AT-1. However, the EA sub-fraction showed little ability on suppression of ROS. Furthermore, we performed an in vivo study using aging mice to be fed with ATE and the sub-fractions followed by immunohistochemical (IHC) staining. The expression of p53 and SA-ß-gal was significantly reduced in several tissue sections, including skin, liver, kidney, and spleen. Taken together, current data demonstrated that A. taiwanensis could suppress cellular senescence in HDFs, and might be used for health care.

Comparison of cofilin­1 and Twist­1 protein expression in human non­small cell lung cancer tissues.

Metastasis is the primary cause of mortality in patients with non­small cell lung cancer (NSCLC). Actin cytoskeletal reorganization is usually accompanied by the epithelial­mesenchymal transition (EMT)­induced invasion and metastasis of cancer cells. In the present study, expression levels of the actin­associated protein cofilin­1 and of the pivotal EMT molecule Twist­1 were determined in NSCLC tissues. Using lung cancer tissue arrays, the identification of 67.4% of tissue spots that exhibited reciprocal levels of cofilin­1 and Twist­1 was achieved by immunohistochemical (IHC) staining. This reciprocal expression pattern was also detected in 21 out of 25 clinicopathological NSCLC tissue sections, and in 10 out of 15 NSCLC cell lines. In addition, high levels of cofilin­1 and low levels of Twist­1 accounted for 80 and 71.5% of the reciprocal expression pattern in tissue arrays and clinicopathological tissue samples, respectively. This pattern was also detected in normal lung tissues, stage I and II lung cancer tissues, and adenocarcinoma subtypes of NSCLC tissues. Although cofilin­1 and Twist­1 were expressed inversely, a positive correlation of these two proteins was present in normal lung tissues and lung tumor tissues. Furthermore, enforced expression of cofilin­1 suppressed the expression level of Twist­1 in NSCLC H1299 cells. An on­line Kaplan­Meier survival analytic tool allowed access to a public microarray dataset with a maximum of 1,926 NSCLC samples. The analysis revealed that high expression levels of both cofilin­1 (CFL1) and Twist­1 (TWIST1) genes were associated with decreased survival of NSCLC patients, notably with regard to the adenocarcinoma subtype. The analysis was conducted using the multivariate Cox regression model. Although the reciprocal association of the expression levels of cofilin­1 and Twist­1 with the survival rate of NSCLC patients requires additional information, it may be a significant indicator of the progression of NSCLC.

Synergistic effects of tetrandrine combined with ionizing radiation on a murine colorectal carcinoma­bearing mouse model.

Tetrandrine (TET), a traditional Chinese clinical agent, has been used for the treatment of many diseases, including cancers. The purpose of the present study was to investigate the combined effects of TET and ionizing radiation (IR) on murine CT26 colorectal adenocarcinoma cells in vitro and in vivo. A CT26 cell line transfected with dual HSV­1 thymidine kinase and firefly luciferase (luc) reporter genes was used. The half­maximal inhibitory concentration (IC50) of TET in CT26/tk­luc cells was ~10 µM. An additive effect was observed after combination of both agents based on a colony formation assay. Apoptosis and cleaved caspase­3 levels were increased significantly in cells after combination treatment, as shown by flow cytometric analysis, DNA fragmentation and western blotting. However, tumor growth inhibition and therapeutic efficacy of TET combined with IR in vivo were identified to be synergistic, as monitored by tumor growth delay time, measured with a digital caliper. A significant inhibition of tumor growth was identified in the combination group compared with the radiation only group. Furthermore, non­invasive bioluminescent imaging (BLI) and gamma scintigraphy were also used to evaluate therapeutic efficacy. Both modalities revealed that the best tumor growth control was under combination treatment among all groups. The present study demonstrated that TET is not only beneficial for chemotherapy, but also has potential as a radiosensitizer for the treatment of cancer.

Arsenic trioxide-mediated suppression of miR-182-5p is associated with potent anti-oxidant effects through up-regulation of SESN2.

Arsenic trioxide (ATO) is a traditional Chinese medicine that can induce oxidative stress for treatment of cancer cells. However, ATO may generate anti-oxidative responses to compromise the cytotoxic effect, but the underlying mechanisms remain unclear. Here we found that ATO could inhibit miR-182-5p expression in patient-derived primary S1 glioblastoma (GBM) cells accompanied by up-regulation of Sestrin-2 (SESN2) mRNA, a known anti-oxidant molecule. This phenomenon was also detected in a U87MG glioma cell line, human lung adenocarcinoma H1299 cell line and A549 cell line. Pretreatment with a free radical scavenger N-acetylcysteine (NAC) reduced the oxidative stress induced by ATO. Concomitantly, ATO mediated suppression of miR-182-5p and enhancement of SESN2 expression were also compromised. The MTT assay further showed that ATO induced cytotoxicity was enhanced by transfection of miR-182-5p mimics. Overexpression of miR-182-5p mimics significantly suppressed the expression of SENS2 and a firefly luciferase reporter gene fused to 3'- untranslated region (UTR) of SESN2 mRNA. Use of ribonucleoprotein immunoprecipitation (RNP-IP), ATO mediated suppression of miR-182-5p led to the stabilization of SESN2 mRNA as a result of Argonaute-2 (AGO2) dependent gene silencing. Furthermore, high expression of miR-182-5p and low expression of SESN2 mRNA tend to be associated with longer survival of glioma or lung cancer patients using public available gene expression datasets and online tools for prediction of clinical outcomes. Taken together, current data suggest that the miR-182-5p/SENS2 pathway is involved in ATO induced anti-oxidant responses, which may be important for the design of novel strategy for cancer treatment.

Combination of Radiofrequency Ablation and Glycated Chitosan as Treatment on a Syngeneic Breast Tumor Model.

AIM: Effects of radiofrequency ablation (RFA) combining immunoadjuvant glycated chitosan (GC) on tumor control and potent cytokine responses were investigated in a syngeneic breast tumor model. MATERIALS AND METHODS: Murine 4T1 breast carcinoma cells harboring the luciferase reporter gene were used to evaluate the tumor growth rate and metastasis in vivo using bioluminescent imaging. Plasma of RFA/GC-treated tumor-bearing mice was collected for ex vivo cytotoxicity analysis and mouse chemokine array assays. RESULTS: Tumor growth and systemic metastasis were suppressed by combined RFA and GC when tumor size reached 300 mm3, not detected, however, when tumor size reached 800 mm3 The survival rate of mice bearing small tumors was also higher than that of large ones after RFA-GC treatment. Plasma extracted from RFA-GC-treated small tumor-bearing mice exhibited cytotoxicity on cultured 4T1 cells. Moreover, reduced tumor growth-related cytokines and increased antitumor-related cytokines were detected in the plasma collected. CONCLUSION: RFA combining GC could control tumor progression with induced potent antitumor cytokine responses.

Combining fisetin and ionizing radiation suppresses the growth of mammalian colorectal cancers in xenograft tumor models.

Fisetin (3,7,3',4'-tetrahydroxyflavone), which belongs to the flavonoid group of polyphenols and is found in a wide range of plants, has been reported to exhibit a number of biological activities in human cancer cells, including antioxidant, anti-inflammatory, antiangiogenic, anti-invasive and antiproliferative effects. Although previous in vitro studies have shown that fisetin treatment increases the apoptotic rate and enhances the radiosensitivity of human colorectal cancer cells, the in vivo effects of fisetin on tumor growth remain unclear. In the present study a murine xenograft tumor model was employed to investigate the therapeutic effects of fisetin in combination with radiation on CT-26 colon cancer cells and human HCT116 colorectal cancer cells. This revealed that intratumoral injection of fisetin significantly suppressed the growth of CT-26 tumors compared with the untreated control group, but had little effect on the growth of HCT116 tumors. However, fisetin in combination with 2-Gy radiation enhanced tumor suppressor activity in murine colon and human colorectal xenograft tumors, as compared with 2-Gy fractionated radiation administered alone for 5 days and fisetin alone. Interestingly, fisetin downregulated the expression of the oncoprotein securin in a p53-independent manner. However, securin-null HCT116 tumors showed only moderate sensitivity to fisetin treatment, and the combination of fisetin and radiation did not significantly suppress securin-null HCT116 tumor growth compared with normal HCT116 tumors. Therefore, the role of securin in mediating the effect of fisetin on colorectal cancer growth warrants further investigation. In conclusion, the results of the current study provide important preclinical data for evaluating the efficacy of fisetin and radiation combination treatment as an adjuvant chemoradiotherapy for human colorectal cancers.

The actin depolymerizing factor (ADF)/cofilin signaling pathway and DNA damage responses in cancer.

The actin depolymerizing factor (ADF)/cofilin protein family is essential for actin dynamics, cell division, chemotaxis and tumor metastasis. Cofilin-1 (CFL-1) is a primary non-muscle isoform of the ADF/cofilin protein family accelerating the actin filamental turnover in vitro and in vivo. In response to environmental stimulation, CFL-1 enters the nucleus to regulate the actin dynamics. Although the purpose of this cytoplasm-nucleus transition remains unclear, it is speculated that the interaction between CFL-1 and DNA may influence various biological responses, including DNA damage repair. In this review, we will discuss the possible involvement of CFL-1 in DNA damage responses (DDR) induced by ionizing radiation (IR), and the implications for cancer radiotherapy.

Enhanced cellular radiosensitivity induced by cofilin-1 over-expression is associated with reduced DNA repair capacity.

PURPOSE: A previous report has indicated that over-expression of cofilin-1 (CFL-1), a member of the actin depolymerizing factor (ADF)/cofilin protein family, enhances cellular radiosensitivity. This study explores the involvement of various DNA damage responses and repair systems in the enhanced cellular radiosensitivity as well as assessing the role of CFL-1 phosphorylation in radiosensitivity. MATERIALS AND METHODS: Human non-small lung cancer H1299 cells harboring a tet-on gene expression system were used to induce exogenous expression of wild-type CFL-1. Colony formation assays were used to determine cell survival after γ-ray exposure. DNA damage levels were determined by Comet assay. DNA repair capacity was assessed by fluorescence-based DNA repair analysis and antibody detection of various repair proteins. The effects of CFL-1 phosphorylation on radiation responses were explored using two mutant CFL-1 proteins, S3D and S3A. Finally, endogenous CFL-1 phosphorylation levels were investigated using latrunculin A (LA), cytochalasin B (CB) and Y27632. RESULTS: When phosphorylatable CFL-1 was expressed, radiosensitivity was enhanced after exposure to γ-rays and this was accompanied by DNA damage. Phosphorylated histone H2AX (γ-H2AX) and p53-binding protein-1 (53BP1) foci, as well as Chk1/2 phosphorylation, were apparently suppressed, although ataxia telangiectasia mutated (ATM) kinase activation was apparently unaffected. In addition, two radiation-induced double-strand break (DSB) repair systems, namely homologous recombination repair (HRR) and non-homologous end joining (NHEJ), were suppressed. Moreover, over-expression of CFL-1 S3D and CFL-1 S3A both enhanced radiosensitivity. However, enhanced radiosensitivity and reduced γ-H2AX expression were only detected in cells treated with LA which increased endogenous phospho-CFL-1, and not in cells treated with Y27632, which dephosphorylates CFL-1. CONCLUSION: CFL-1 over-expression enhances radiosensitivity and this is associated with reduced DNA repair capacity. Although phosphorylated CFL-1 seems to be involved in radiosensitivity, further studies are required to address the importance of CFL-1 activity to the regulation of radiosensitivity.

The effect of diminished osteogenic signals on reduced osteoporosis recovery in aged mice and the potential therapeutic use of adipose-derived stem cells.

Adipose-derived stem cells (ADSCs) have been shown to be pluoripotent and explored for their usage in tissue engineering. Previously, we have established a cell-based approach comprised of platelet-enriched plasma and osteo-progenitor cells for treating osteoporosis in an ovariectomized-senescence-accelerated mice (OVX-SAMP8) model. In the present study, we intend to explore the feasibility of using ADSCs as a cell-based therapeutic approach for treating osteoporosis, and to examine the effects of aging on the pluoripotency of ADSCs and the efficiency of bone formation both in vitro and in vivo. Flow cytometry was used to characterize ADSCs isolated from young and aged female SAMP8 mice and showed that the highly positive expression of surface markers such as CD44 and CD105 and negative for CD34 and CD45. Therefore, to compare the aging effects on the growth kinetics and differentiation potential of young and aged ADSCs, we found that there was a significant decline in both the proliferation rate (approximately 13.3%) and osteo-differentiation potential in aged ADSC. Subsequently, young and aged ADSCs were transplanted into the bone marrow of osteoporotic mice (OVX-SAMP8) to evaluate their bone formation ability. ADSC transplants were shown effective in restoring bone mineral density in the right/left knees, femurs and spine, 4 months post-transplantation; mice which received young ADSC transplants showed significantly higher bone regeneration (an average of 24.3% of improved BMD) over those received aged ADSCs. In conclusion, these findings showed that aging impedes osteoporosis-ameliorating potential of ADSC by diminishing osteogenic signal, and that ADSC could be used as a potential cell-based therapy for osteoporosis.

Involvement of p53 R72P polymorphism in the association of MDM2-SNP309 with breast cancer.

The allelic variant MDM2-SNP309 (T>G) has been suggested to influence cancer development, but the clinical correlation between the risk allele and breast cancer remains controversial. The genetic background and the ethnicity of selected subgroups may influence the power of these risk genotypes. In this study, we investigated whether MDM2-SNP309 is associated with p53 R72P genetic polymorphism for the risk of breast cancer development in Asian Taiwanese, which has not been well-studied in this regard. Two hundred and fifty-five patients and 324 cancer-free controls were included, and we found that the MDM2-SNP309 TG and GG genotypes displayed marginally increased risks of breast cancer (GG vs. TT: OR = 1.7, 95% CI = 0.93 to 3.09; TG + TT vs. TT: OR = 1.57, 95% CI = 0.98 to 2.56). The breast cancer risk associated with MDM2-SNP309 was enhanced after stratification for the homozygous GG genotype at p53 codon 72 representing the Arg form of this genotype (GG vs. TT: OR = 3.7, 95% CI = 1.144 to 12.02; TG + GG vs. TT: OR = 2.7, 95% CI = 1.027 to 6.895). Also, the median age at diagnosis of patients with MDM2-SNP309 GG increased from 4 years earlier to 9 years earlier than TT patients after stratification for the GG genotype at p53 codon 72. Moreover, the G-allele of MDM2-SNP309 exhibited a stronger capacity than the T-allele to drive the full-length P2 promoter of the MDM2 gene in several human cell lines, suggesting that the association between MDM2-SNP309 and breast cancer is likely multifactorial rather than due to inconsistent gene expression in different cancer sources.

Association between MDM2-SNP309 and hepatocellular carcinoma in Taiwanese population.

AIM: To investigate the risk association and compare the onset age of hepatocellular carcinoma (HCC) patients in Taiwan with different genotypes of MDM2-SNP309. METHODS: We analyzed MDM2-SNP309 genotypes from 58 patients with HCC and 138 cancer-free healthy controls consecutively. Genotyping of MDM2-SNP309 was conducted by restriction fragment length polymorphism assay. RESULTS: The proportion of homozygous MDM2-SNP309 genotype (G/G) in cases and cancer-free healthy controls was similar (17.2% vs 16.7%). Multivariate analysis showed that the risk of G/G genotype of MDM2-SNP309 vs wild-type T/T genotype in patients with HCC was not significant (OR = 1.265, 95% CI = 0.074-21.77) after adjustment for sex, hepatitis B or C virus infection, age, and cardiovascular disease/diabetes. Nevertheless, there was a trend that GG genotype of MDM2-SNP309 might increase the risk in HCC patients infected with hepatitis virus (OR = 2.568, 95% CI = 0.054-121.69). Besides, the homozygous MDM2-SNP309 genotype did not exhibit a significantly earlier age of onset for HCC. CONCLUSION: Current data suggest that the association between MDM2-SNP309 GG genotype and HCC is not significant, while the risk may be enhanced in patients infected by hepatitis virus in Taiwan.

Results based on 124 cases of breast cancer and 97 controls from Taiwan suggest that the single nucleotide polymorphism (SNP309) in the MDM2 gene promoter is associated with earlier onset and increased risk of breast cancer.

BACKGROUND: It has been suggested that the single nucleotide polymorphism 309 (SNP309, T -> G) in the promoter region of the MDM2 gene is important for tumor development; however, with regards to breast cancer, inconsistent associations have been reported worldwide. It is speculated that these conflicting results may have arisen due to different patient subgroups and ethnicities studied. For the first time, this study explores the effect of the MDM2 SNP309 genotype on Taiwanese breast cancer patients. METHODS: Genomic DNA was obtained from the whole blood of 124 breast cancer patients and 97 cancer-free healthy women living in Taiwan. MDM2 SNP309 genotyping was carried out by restriction fragment length polymorphism (RFLP) assay. The multivariate logistic regression and the Kaplan-Meier method were used for analyzing the risk association and significance of age at diagnosis among different MDM2 SNP309 genotypes, respectively. RESULTS: Compared to the TT genotype, an increased risk association with breast cancer was apparent for the GG genotype (OR = 3.05, 95% CI = 1.04 to 8.95), and for the TG genotype (OR = 2.12, 95% CI = 0.90 to 5.00) after adjusting for age, cardiovascular disease/diabetes, oral contraceptive usage, and body mass index, which exhibits significant difference between cases and controls. Furthermore, the average ages at diagnosis for breast cancer patients were 53.6, 52 and 47 years for those harboring TT, TG and GG genotypes, respectively. A significant difference in median age of onset for breast cancer between GG and TT+TG genotypes was obtained by the log-rank test (p = 0.0067). CONCLUSION: Findings based on the current sample size suggest that the MDM2 SNP309 GG genotype may be associated with both the risk of breast cancer and an earlier age of onset in Taiwanese women.