Increased radiosensitivity of colorectal tumors with intra-tumoral injection of low dose of gold nanoparticles.

The potential of gold nanoparticles (GNPs) as radiosensitizers for the treatment of malignant tumors has been limited by the large quantities of GNPs that must be administered and the requirement for low-energy X-ray irradiation to optimize radiosensitization. In this study, we enhance the radiosensitivity of HCT116 human colorectal cells with tiopronin-coated GNPs (Tio-GNPs) combined with a low-energy X-ray (26 keV effective energy) source, similar to the Papillon 50 clinical irradiator used for topical irradiation of rectal tumors. Sensitizer enhancement ratios of 1.48 and 1.69 were measured in vitro, when the HCT116 cells were incubated with 0.1 mg/mL and 0.25 mg/mL of Tio-GNPs, respectively. In nude mice bearing the HCT116 tumor, intra-tumoral (IT) injection of Tio-GNPs allowed a 94 times higher quantity of Tio-GNPs to accumulate than was possible by intravenous injection and facilitated a significant tumor response. The time following irradiation, for tumors growing to four times their initial tumor volume (4Td) was 54 days for the IT injection of 366.3 µg of Tio-GNPs plus 10 Gy, compared to 37 days with radiation alone (P=0.0018). Conversely, no significant improvement was obtained when GNPs were injected intravenously before tumor irradiation (P=0.6547). In conclusion, IT injection of Tio-GNPs combined with low-energy X-rays can significantly reduce the growth of colorectal tumors.

[ID1 suppress the apoptosis of HCT116 cells induced by chemotherapeutic drugs and ultraviolet radiation].

OBJECTIVE: To investigate the changes of ID1 expression in tumor cells treated with etoposide, cisplatin and ultraviolet (UV) irradiation, and explore the effect of ID1 on chemotherapeutic drug- and UV-induced apoptosis. METHODS: In the present study, upon onset of apoptosis induced by various kinds of inducers such as etoposide, cisplatin and UV irradiation, the expression level of ID1 was detected by Western blot and real-time PCR. We also analyzed the half-life of ID1 protein and stability of ID1 mRNA respectively by cycloheximide inhibition test and RT-PCR. Annexin-V assay was carried out to evaluate the contribution of ID1 protein to chemotherapeutic drug- and UV-induced apoptosis. RESULTS: ID1 expression presented a profound down-regulation in the HCT116 cells treated with etoposide, cisplatin and UV irradiation(P<0.05 for all). The apoptosis in the UV irradiation group, cisplatin group, etoposide group was (58.70±1.55)%, (35.80±0.92)% and (21.00±0.72)%, respectively, significantly higher than that of the control group(1.10±0.07)%, (1.20±0.13)% and (3.50±0.23)% (P<0.05 for all). Upon etoposide treatment, ID1 expression level was decreased via induction of mRNA instability, but not the protein degradation changes. Additionally, ectopic expression of ID1 in the HCT116 cells alleviated etoposide-, cisplatin- and UV-induced apoptosis. The results of flow eytometry revealed that the percentage of apoptotic cells in the ID1 group under the treatment of etoposide, cisplatin and UV irradiation was (23.80±0.82)%, (17.80±1.34)% and (13.40±0.53)%, respectively, significantly lower than that in the empty vector group (41.10±1.61)%, (30.40±2.67)% and (22.50±3.47)% (P<0.05 for all). CONCLUSIONS: These observations indicate that the treatment with etoposide reduces the amount of ID1 by induction of mRNA instability, and exogenously introduced ID1 protects cells against etoposide-, cisplatin- and UV irradiation-induced apoptosis. Inhibition of the ID1 bioactivity may become a new strategy in cancer treatment.

125I Seeds Radiation Induces Paraptosis-Like Cell Death via PI3K/AKT Signaling Pathway in HCT116 Cells.

125I seeds brachytherapy implantation has been extensively performed in unresectable and rerecurrent rectal carcinoma. Many studies on the cancer-killing activity of 125I seeds radiation mainly focused on its ability to trigger apoptosis, which is the most well-known and dominant type of cell death induced by radiation. However our results showed some unique morphological features such as cell swelling, cytoplasmic vacuolation, and plasma membrane integrity, which is obviously different to apoptosis. In this study, clonogenic proliferation was carried out to assay survival fraction. Transmission electron microscopy was used to analyze ultrastructural and evaluate morphologic feature of HCT116 cells after exposure to 125I seeds radiation. Immunofluorescence analysis was used to detect the origin of cytoplasmic vacuoles. Flow cytometry analysis was employed to detect the size and granularity of HCT116 cells. Western blot was performed to measure the protein level of AIP1, caspase-3, AKT, p-Akt (Thr308), p-Akt (Ser473), and ß-actin. We found that 125I seeds radiation activated PI3K/AKT signaling pathway and could trigger paraptosis-like cell death. Moreover, inhibitor of PI3K/AKT signaling pathway could inhibit paraptosis-like cell death induced by 125I seeds radiation. Our data suggest that 125I seeds radiation can induce paraptosis-like cell death via PI3K/AKT signaling pathway.

Enhancement of p53-mutant human colorectal cancer cells radiosensitivity by flavonoid fisetin.

PURPOSE: The aim of this study was to investigate whether fisetin is a potential radiosensitizer for human colorectal cancer cells, which are relatively resistant to radiotherapy. METHODS AND MATERIALS: Cell survival was examined by clonogenic survival assay, and DNA fragmentation was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. The effects of treatments on cell cycle distribution and apoptosis were examined by flow cytometry. Western blot analysis was performed to ascertain the protein levels of gamma-H2AX, phospho-Chk2, active caspase-3, PARP cleavage, phospho-p38, phospho-AKT, and phospho-ERK1/2. RESULTS: Fisetin pretreatment enhanced the radiosensitivity of p53-mutant HT-29 human colorectal cancer cells but not human keratocyte HaCaT cells; it also prolonged radiation-induced G(2)/M arrest, enhanced radiation-induced cell growth arrest in HT-29 cells, and suppressed radiation-induced phospho-H2AX (Ser-139) and phospho-Chk2 (Thr-68) in p53-mutant HT-29 cells. Pretreatment with fisetin enhanced radiation-induced caspase-dependent apoptosis in HT-29 cells. Fisetin pretreatment augmented radiation-induced phosphorylation of p38 mitogen-activated protein kinase, which is involved in caspase-mediated apoptosis, and SB202190 significantly reduced apoptosis and radiosensitivity in fisetin-pretreated HT-29 cells. By contrast, both phospho-AKT and phospho-ERK1/2, which are involved in cell proliferation and antiapoptotic pathways, were suppressed after irradiation combined with fisetin pretreatment. CONCLUSIONS: To our knowledge, this study is the first to provide evidence that fisetin exerts a radiosensitizing effect in p53-mutant HT-29 cells. Fisetin could potentially be developed as a novel radiosensitizer against radioresistant human cancer cells.

Depletion of securin induces senescence after irradiation and enhances radiosensitivity in human cancer cells regardless of functional p53 expression.

PURPOSE: Radiotherapy is one of the best choices for cancer treatment. However, various tumor cells exhibit resistance to irradiation-induced apoptosis. The development of new strategies to trigger cancer cell death besides apoptosis is necessary. This study investigated the role of securin in radiation-induced apoptosis and senescence in human cancer cells. METHODS AND MATERIALS: Cell survival was determined using clonogenic assays. Western blot analysis was used to analyze levels of securin, caspase-3, PARP, p53, p21, Rb, gamma-H2AX, and phospho-Chk2. Senescent cells were analyzed using a beta-galactosidase staining assay. A securin-expressed vector (pcDNA-securin) was stably transfected into securin-null HCT116 cells. Securin gene knockdown was performed by small interfering RNA and small hairpin RNA in HCT116 and MDA-MB-231 cells, respectively. RESULTS: Radiation was found to induce apoptosis in securin wild type HCT116 cells but induced senescence in securin-null cells. Restoration of securin reduced senescence and increased cell survival in securin-null HCT116 cells after irradiation. Radiation-induced gamma-H2AX and Chk2 phosphorylation were induced transiently in securin-wild-type cells but exhibited sustained activation in securin-null cells. Securin gene knockdown switches irradiation-induced apoptosis to senescence in both HCT116 p53-null and MDA-MB-231 cells. CONCLUSIONS: Our results demonstrated that the level of securin expression plays a determining role in the radiosensitivity and fate of cells. Depletion of securin impairs DNA repair after irradiation, increasing DNA damage and promoting senescence in the residual surviving cells regardless of functional p53 expression. The knockdown of securin may contribute to a novel radiotherapy protocol for the treatment of human cancer cells that are resistant to irradiation.

CD133 expression is not selective for tumor-initiating or radioresistant cell populations in the CRC cell lines HCT-116.

BACKGROUND AND PURPOSE: CD133 is controversially discussed as putative (surrogate) marker for cancer stem/tumor-initiating cell populations (CSC/TIC) in epithelial tumors including colorectal carcinomas (CRCs). We studied CD133 expression in established CRC cell lines and examined in vitro behavior, radioresponse and in vivo tumor formation of CD133(+/-) subpopulations of one cell line of interest. MATERIALS AND METHODS: Ten CRC cell lines were analyzed for CD133 expression using flow cytometry and Western blotting. CD133+ and CD133(-) HCT-116 subpopulations were separated by FACS and studied in 2-D and 3-D culture and colony formation assays after irradiation. Subcutaneous xenograft formation was monitored in NMRI (nu/nu) mice. RESULTS AND CONCLUSIONS: CRC cell lines could be classified into three groups: (i) CD133(-), (ii) CD133+ and (iii) those with two distinct CD133+ and CD133(-) subpopulations. Isolated CD133(+/-) HCT-116 subpopulations were studied relative to the original fraction. No difference was found in 2-D growth, spheroid formation or radioresponse in vitro. Also, tumor formation and growth rate did not differ for the sorted subpopulations. However, a subset of xenografts originated from CD133(-) HCT-116 showed a striking enrichment in the CD133+ fraction. Our data show that CD133 expression is not selective for sphere forming, tumor-initiating or radioresistant subpopulations in the HCT-116 CRC cell lines. This implies that CD133 cannot be regarded as a CSC/TIC marker in all CRC cell lines and that functional measurements of tumor formation have to generally accompany CSC/TIC-directed mechanistic or therapeutic studies.

Celecoxib induced tumor cell radiosensitization by inhibiting radiation induced nuclear EGFR transport and DNA-repair: a COX-2 independent mechanism.

PURPOSE: The purpose of the study was to elucidate the molecular mechanisms mediating radiosensitization of human tumor cells by the selective cyclooxygenase (COX)-2 inhibitor celecoxib. METHODS AND MATERIALS: Experiments were performed using bronchial carcinoma cells A549, transformed fibroblasts HH4dd, the FaDu head-and-neck tumor cells, the colon carcinoma cells HCT116, and normal fibroblasts HSF7. Effects of celecoxib treatment were assessed by clonogenic cell survival, Western analysis, and quantification of residual DNA damage by gammaH(2)AX foci assay. RESULTS: Celecoxib treatment resulted in a pronounced radiosensitization of A549, HCT116, and HSF7 cells, whereas FaDu and HH4dd cells were not radiosensitized. The observed radiosensitization could neither be correlated with basal COX-2 expression pattern nor with basal production of prostaglandin E2, but was depended on the ability of celecoxib to inhibit basal and radiation-induced nuclear transport of epidermal growth factor receptor (EGFR). The nuclear EGFR transport was strongly inhibited in A549-, HSF7-, and COX-2-deficient HCT116 cells, which were radiosensitized, but not in FaDu and HH4dd cells, which resisted celecoxib-induced radiosensitization. Celecoxib inhibited radiation-induced DNA-PK activation in A549, HSF7, and HCT116 cells, but not in FaDu and HH4dd cells. Consequentially, celecoxib increased residual gammaH2AX foci after irradiation, demonstrating that inhibition of DNA repair has occurred in responsive A549, HCT116, and HSF7 cells only. CONCLUSIONS: Celecoxib enhanced radiosensitivity by inhibition of EGFR-mediated mechanisms of radioresistance, a signaling that was independent of COX-2 activity. This novel observation may have therapeutic implications such that COX-2 inhibitors may improve therapeutic efficacy of radiation even in patients whose tumor radioresistance is not dependent on COX-2.