Taraxasterol enhanced bladder cancer cells radiosensitivity via inhibiting the COX-2/PGE2/JAK2/STAT3/MMP pathway.

PURPOSE: Radiotherapy with bladder preservation is highly acceptable among patients bearing bladder cancer (BCa), but the occurrence of secondary tolerance (ARR) during treatment is one of the important reasons for the failure of clinical radiotherapy. COX-2 has been frequently reported to be highly expressed and associated with radio-resistance in various cancers. In this study, the feasibility of Taraxasterol (Tara) as a radiosensitizer was investigated, and the target effect of Tara on COX-2 and its underlying mechanism were explored. METHODS AND MATERIALS: The toxicity of Tara toward BCa cells was detected with the MTT method and cells in response to IR or Tara + IR were compared by clone formation assay. Next, a small RNA interference system (siRNA) was employed to decrease endogenous COX-2 expression in BCa cells, and the stem cell-like features and motion abilities of BCa cells under different treatments were investigated using microsphere formation and transwell chamber assay, respectively. Meanwhile, the expression of a series of inflammation-related molecules and stem cell characteristic molecules was determined by qRT-PCR, western blot and ELISA method. In vivo studies, BCa cells were subcutaneously injected into the right flank of each male mouse. Those mice were then grouped and exposed to different treatment: Tara, IR, IR + Tara and untreated control. The volumes of each tumor were measured every two days and target proteins were detected with immunohistochemical (IHC) staining. RESULTS: The results show that COX-2 decline, due to COX-2 knocking-down or Tara treatment, could greatly enhance BCa cells' radiosensitivity and significantly decrease their migration, invasion and microsphere formation abilities, companied with the reduce of JAK2, phos-STAT3, MMP2 and MMP9 expression. However, Tara could not further reduce the expression of an above molecule of cells in COX-2-deficient BCa cells. Correspondingly, Tara treatment could not further enhance those siCOX-2 BCa cells response to IR. CONCLUSIONS: Our data support that Tara can improve the radiosensitivity of BCa cells by targeting COX-2/PGE2. The mechanism may involve regulating STAT3 phosphorylation, DNA damage response protein activation, and expression of MMP2/MMP9.

DAB2IP-knocking down resulted in radio-resistance of breast cancer cells is associated with increased hypoxia and vasculogenic mimicry formation.

PURPOSE: As a part of breast-conserving therapy (BCT), postoperative radiotherapy is one of the main means to improve the clinical efficacy of breast cancer (BCa). However, ionizing radiation (IR) may induce BCa cells to develop radioresistance, which causes tumor recurrence and metastasis after treatment. Recently, DOC-2/DAB2 interactive protein (DAB2IP) has been reported often down-regulated in a variety of cancers and is related to tumor tolerance to radiotherapy. In this study, BCa cell lines were introduced to study how DAB2IP deficient influenced BCa cell radiosensitivity in vitro and in vivo and discuss the possible mechanism. METHODS AND MATERIALS: Small RNA interference system (siRNA) was employed to decrease DAB2IP expression in two BCa cell lines, MDA-MB-231 and 4T1. Cells in response to IR or antineoplastics were detected by clone formation assay or MTT method, respectively. For in vivo studies, siDAB2IP or siControl cells were subcutaneously injected into the right flank of each female mouse. Sphere formation assay, soft agar colony anchoring assay and in vivo tumorigenesis assay were implemented to examine the stem cell-like features of BCa cells. Tube formation assay as well as immunofluorescence assay (IFA) were respectively applied to determine the angiogenesis of tumor cells in vitro and in vivo. The expression of a series of angiogenesis-related molecules was analyzed by qRT-PCR, western blot and IFA. RESULTS: It was observed that the downregulation of DAB2IP could significantly improve the clone formation ability of BCa cells, reduce their sensitivity to radiation and chemotherapy drugs, enhance their migration and invasion abilities and increase their stemness characteristics. It was also noted that either DAB2IP-knocking down or treated with the conditioned medium from DAB2IP-deficient BCa cells could promote the tube-forming ability of the endothelial cell. Similarly, in vivo studies showed that tumors developed from siDAB2IP BCa cells had higher tumor microvascular density (MVD) and more severe oxygen deficiency than that in DAB2IP- sufficient tumors. Meanwhile, Knock-down of DAB2IP inhibited vascular maturation and promoted the formation of vasculogenic mimicry (VM) in BCa tissues. Down-regulation of STAT3 could enhance siDAB2IP cells sensitivity to IR, accompanied by the decrease of VEGF expression. CONCLUSIONS: Our data support that loss of DAB2IP confers radio-resistance of BCa could be due to increased hypoxia, inhibited vascular maturation and promoted VM formation. STAT3 inhibition could be a potential way to overcome such DAB2IP-deficient induced tolerance in BCT.

PD-1 Inhibitor Enhanced Radiosensitivity by Reactivating T Cells and Inducing G2/M Phase Arrest in Esophageal Squamous Cell Carcinoma.

Radiotherapy is a main treatment for esophageal squamous cell carcinoma (ESCC), but radioresistance leads to treatment failure ultimately. The combination of radiotherapy and PD-1 inhibitors showed significant antitumor effects. Our study showed that high-immune score, IFNG and CD8A level were associated with a low-radiosensitivity index (RSI) in the TCGA-ESCC cohort. And blocking PD-1 promoted exhausted T cells proliferation and IFN-γ expression. PD-1 inhibitor-reactivated T cells promoted G2/M-phase arrest, apoptosis and impaired DNA damage in radioresistant cells in an IFN-γ-dependent manner. Our study showed PD-1 inhibitors promote radiosensitivity though enhancing exhausted T cells expansion and IFN-γ expression, and highlights that neoadjuvant anti-PD-1 therapy and radiotherapy could offer an optimum strategy for improving cancer patients' outcome.

EZH2 targeting to improve the sensitivity of acquired radio-resistance bladder cancer cells.

Among the many treatments for Bladder cancer (BCa) patients, radiotherapy is an effective way to preserve the bladder. However, as the frequency of irradiation increases, the tumor cells appear "acquired radio-resistance" (ARR) and loss the sensitivity to radiotherapy. To explore the molecular mechanism of ARR, two BCa cell lines, 5637 and T24, were enrolled here and their ARR counterparts, 5637R and T24R, were obtained by exposure to γ-ray of 2 Gy for 30 times. Compared to parental cells, ARR cells have significantly enhanced stem cell-like phenotype, robust DNA damage repair capabilities and elevated expression of zeste homolog 2 (EZH2). Decreasing EZH2 expression, both parental and ARR cells exhibited reduced abilities of forming microsphere and repairing DNA damage, but enhanced cells radio-sensitivity and intracellular autophagy compared to untreated cells. Down-regulation the expression of EZH2 induced an increasing of both LC3 and P62 in parental cells, while in ARR cells, only LC3 increased upon EZH2 reduction. On the other hand, UNC1999 treatment caused the increasing of LC3B and P62 in all cells, suggested that siEZH2 and UNC1999 affect ARR cells autophagy through different mechanisms. In vivo study showed that pre-treated with UNC1999 greatly enhanced T24R cells sensitivity to IR, and knocking down the expression of EZH2 significantly suppressed the tumor growth. Combined with bioinformatics data analysis, we speculate that EZH2 is an important biomolecule linking the diagnosis, radiotherapy and prognosis of BCa. EZH2 targeted therapy may be an effective way to overcome ARR of BCa, and is worthy of in-depth study.

Down-regulation of autophagy-associated protein increased acquired radio-resistance bladder cancer cells sensitivity to taxol.

BACKGROUND: As a bladder-preserving therapy, radiation therapy (RT) has been widely used in the treatment of bladder cancer (BCa) and made great progress in the past few decades. However, some BCa patients have low RT responsiveness and local recurrence rate after RT could reach 50%. Acquired radio-resistance (ARR) is one of the important reasons for the failure of RT. Unfortunately, these ARR cells also lack sensitivity to chemotherapy and cause tumor recurrence and metastasis. PURPOSE: To build ARR-phenotype BCa cell model, discuss the possible molecular mechanism of ARR and find effective target molecules to overcome ARR. MATERIALS AND METHODS: Five thousand six hundred and thirty-seven cells were subjected 30 times to 2 Gy of γ-rays and the surviving cells were called 5637R. Colony formation and MTT assay were applied to evaluate cells sensitivity to ionizing radiation (IR) and anti-neoplastic agents, respectively. Cells abilities of migration and invasion were determined using transwell method. Quantitative real-time polymerase chain reaction (RT-qPCR) and western blot (WB) were respectively utilized to compare the difference of gene and protein expression between 5637 and 5637R cells. Molecule inhibitors and small interfering RNA (siRNA) systems were employed to decrease the expression of target proteins, respectively. RESULTS: BCa cells survived from fractionated irradiation (FI) exhibited tolerance to both IR and chemotherapy drugs. These ARR cells (5637R) had elevated migration and invasion abilities, accompanied by increased expression of epithelial mesenchymal transition (EMT)-related transcription factors (ZEB1/Snail/Twist). Moreover, 5637R cells showed enhanced cancer stem cell (CSC)-like characteristics with activated KMT1A-GATA3-STAT3 circuit, a newly reported self-renewal pathway of human bladder cancer stem cell (BCSC). Combined with Kaplan-Meier's analysis, we speculated that GATA3/MMP9/STAT3 could be an effective molecular panel predicting poor prognosis of BCa. In order to enhance the sensitivity of resistant cells to radiation, we introduced ERK inhibitor (FR 180204) and STAT3 inhibitor (S3I-201). However, both of them could not enhance ARR cells response to IR. On the other hand, siRNAs were respectively implemented to inhibit the expression of endogenous Beclin1 and Atg5, two important autophagy-related genes, in BCa cells, which significantly increased 5637R cells death upon taxol exposing. Similarly, chloroquine (CQ), a classic autophagy inhibitor, enhanced the cytotoxicity of taxol only on 5637R cells. CONCLUSIONS: Long-term FI treatment is an effective method to establish the ARR-phenotype BCa cell model, by enriching BCSCs and enhancing cells migration and invasion. Both inhibiting the expression of autophagy-related proteins and using autophagy inhibitor can increase the sensitivity of ARR cells to taxol, suggesting that autophagy may play an important role in ARR cells chemical tolerance.

STAT3 enhances radiation-induced tumor migration, invasion and stem-like properties of bladder cancer.

Bladder cancer (BCa) is the most common cancer of the human urinary system, and is associated with poor patient prognosis and a high recurrence rate. Cancer stem cells (CSCs) are the primary cause of tumor recurrence and metastasis, possessing self­renewal properties and resistance to radiation therapy. Our previous studies indicated that phosphorylated signal transduction and transcription activator 3 (STAT3) may be a potential biomarker to predict radiation tolerance and tumor recurrence in patients with BCa, following conventional radiotherapy. The aim of the present study was to investigate the underlying mechanism of STAT3 in the radio­resistance of BCa cells. It was found that fractionated irradiation promoted the activation of two STAT3­associated CSCs signaling pathways in BCa cells, namely suppressor of variegation 3­9 homolog 1/GATA binding protein 3/STAT3 and Janus kinase 2/STAT3. Surviving cells exhibited elevated migratory and invasive abilities, enhanced CSC­like characteristics and radio­resistance. Furthermore, knockdown of STAT3 expression or inhibition of STAT3 activation markedly decreased the self­renewal ability and tumorigenicity of radiation­resistant BCa cells. Kaplan­Meier analysis revealed that decreased STAT3 mRNA levels were associated with increased overall survival times in patients with BCa. Taken together, these data indicated that STAT3 may be an effective therapeutic target for inhibiting the progression, metastasis and recurrence of BCa in patients receiving radiotherapy.

Long term exposure to γ­rays induces radioresistance and enhances the migration ability of bladder cancer cells.

In Western countries, bladder cancer (BCa) is one of the most common types of malignancy, with highest incidence rate among urinary system malignancies. At present, as a strategy to preserve the quality and function of the bladder tissues, external­beam radiotherapy has been applied to an increasing number of patients with BCa, particularly the elderly and those in poor health. The onset of acquired radioresistance (ARR) during radiotherapy notably reduces the effectiveness of radiotherapy; however, the cause of ARR is unclear and remains untreatable. In order to investigate the mechanism of ARR, clinical fractionated irradiation was mimicked in vitro to obtain radioresistant BCa cells, 5637R, in the present study. Compared with the parental cell line, 5637R cells exhibited an increased distribution of S phase cells, enhanced migration ability and elevated levels of phosphorylated­STAT3, as determined using flow cytometry, cell migration analysis and western blotting, respectively. Furthermore, 5637R cells exhibited notable increases in extracellular signal­regulated kinase activation in response to ionizing radiation (IR) compared with 5637 cells. In addition, 5637R cells showed an early G2/M arrest within 8 h post­IR (18.23±3.85 vs. 6.42±3.82%, IR cells vs. control cells), whereas 5637 cells were arrested in G2/M phase 24 h post­IR (19.26±2.21 vs. 12.36±4.45%, IR cells vs. control cells). Overall, the present study suggested that phosphorylated STAT3 may be a potential biomarker to predict radioresistance and tumor recurrence in patients with BCa following conventional radiotherapeutic intervention. In addition, co­treatment with ERK inhibitor may be a viable approach to increase the anticancer efficacy of radiotherapy in patients with ARR; however, further investigation is required.

ATM mediates DAB2IP-deficient bladder cancer cell resistance to ionizing radiation through the p38MAPK and NF-κB signaling pathway.

Although surgery remains the standard therapy for the treatment of bladder cancer (BCa), the data from previous clinical studies suggest that there is an increase in the number of patients with a preference for bladder preservation strategies, including radiotherapy, to improve their life quality. Our preliminary results showed that disabled homolog 2 interactive protein (DAB2IP), a putative tumor suppressor gene, is often downregulated in BCa with a radioresistant phenotype. Subsequent investigations revealed that elevated expression of ataxia­telangiectasia mutated (ATM) induced by DAB2IP­knockdown may be the key event in BCa cell resistance to ionizing radiation (IR). However, how ATM is involved in the survival of DAB2IP­deficient cells exposed to IR remains to be fully elucidated. The present study knocked down the expression of ATM in DAB2IP­deficient BCa cells using RNA interference technology. Activation of mitogen­activated protein kinase (MAPK) and nuclear factor­κB (NF­κB) signaling pathways were detected by western blot analysis and immunofluorescence assay, respectively. It was demonstrated that knockdown of ATM enhanced the response of DAB2IP­deficient BCa cells to IR, which may have resulted from delayed DNA double­strand break repair kinetics, compromised nuclear factor­κB translocation, inhibited phosphorylation of p38 and the induced activation of c­Jun N­terminal kinase. Taken together, these findings suggested that ATM may be an effective target in the radiotherapy of patients with DAB2IP-deficient BCa.

Upregulating DAB2IP expression via EGR-1 inhibition, a new approach for overcoming fractionated-irradiation-induced cross-tolerance to ionizing radiation and mitomycin C in tumor cells.

PURPOSE: To evaluate the effect of fractionated irradiation (FI) on tumor cells' sensitivity to ionizing radiation (IR) and antineoplastic drugs, and examine the potential of early growth response-1 (EGR-1) inhibition to sensitize tumor cells to IR. MATERIALS AND METHODS: PC3 and HepG2 cells were subjected 10 times to γ-rays at 2 Gy. The surviving cells were named PC3/R and HepG2/R, respectively. The cells' sensitivity to irradiation and chemotherapeutic drugs, including cisplatin (PT), doxorubicin (DOX), mitomycin C (MMC) and 5-fluorouracil (5-FU), were identified by colony formation assay and MMT method, respectively. Quantitative real-time polymerase chain reaction (RT-qPCR) analysis was utilized to compare the difference of gene expression between radioresistant cells and parental cells. The small interfering RNA system was implemented to inhibit endogenous EGR-1 expression in radiation-resistant cells. Western blot was employed to identify the possible mechanism by which EGR-1 regulates cells' radiosensitivity. RESULTS: FI induced cross-resistant to IR and MMC in tumor cells. Along with the reduction of ovarian cancer-2/disabled homolog 2 (DOC-2/DAB2) interactive protein (DAB2IP) expression, EGR-1 gene was upregulated in FI-treated cells. On the other hand, downregulation of EGR-1 gene expression sensitized radioresistant cells to IR accompanied by DAB2IP overexpression and STAT3 inactivation. In addition, NF-κB inhibitor, BAY11-7082 enhanced resistant cells' radiosensitivity and chemosensitivity. CONCLUSIONS: Conventionally FI has a higher risk of forming acquired radioresistance (ARR) in vitro. EGR-1 gene-targeted drug design could be an effective strategy to overcome DAB2IP-dysregulation-induced ARR in tumor patients.

Decreased DAB2IP gene expression, which could be induced by fractionated irradiation, is associated with resistance to γ­rays and α­particles in prostate cancer cells.

External beam radiation therapy, alone or combined with androgen deprivation, is a well­established treatment for prostate cancer (PCa). However, not all patients benefit from radiotherapy due to congenital or acquired radioresistance. The preliminary results of the present study indicated that the loss of disabled homolog 2 interactive protein (DAB2IP) expression in PCa and normal prostate epithelia results in the resistance to γ­rays. To further explore the association between DAB2IP and ionizing radiation (IR), PCa cells were fractionally irradiated 12 times with 2 Gy of γ­rays and the change in DAB2IP mRNA expression was monitored. Notably, along with a continuous reduction of DAB2IP expression levels, increased expression levels of ataxia­telangiectasia mutated (ATM) was observed in IR­treated cells. In order to improve the sensitivity of DAB2IP­deficient cells to IR, α­particles, a type of high linear energy transfer radiation and KU55933, an ATM inhibitor, were used in the current study. It was determined that α­particle irradiations were more effective than γ­rays on cells expressing expected and decreased levels of DAB2IP. However, cells with a dysfunctional DAB2IP gene were resistant to α­particle irradiation. Treatment with KU55933 did not enhance cell sensitivity to α­irradiation. Therefore, this suggested that DAB2IP downregulation induced by radiotherapy may be associated with acquired radioresistance in patients with PCa.

The ATM inhibitor KU55933 sensitizes radioresistant bladder cancer cells with DAB2IP gene defect.

PURPOSE: Our preliminary results showed that differentially expressed in ovarian cancer-2/disabled homolog 2 (DOC-2/DAB2) interactive protein (DAB2IP), a putative tumor suppressor gene, is down-regulated in bladder cancer (BCa) with aggressive phenotypes. In this study, we investigated how DAB2IP knockdown influenced BCa cell response to ionizing radiation (IR) and discussed possible ways to enhance cell radiosensitivity. METHODS AND MATERIALS: The small interfering RNA (siRNA) system was implemented to inhibit endogenous DAB2IP expression in two human BCa cell lines, T24 and 5637. Cell sensitivity to IR alone or combined treatment was measured by a colony formation assay (CFA). Western blot was used to determine the phosphorylation levels of ataxia-telangiectasia mutated (ATM), catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) and related DNA damage repair (DDR) proteins. Immunofluorescence as well as a flow cytometry assay were employed to detect DNA double-strand break (DSB) repair and cell cycle distribution, respectively. RESULTS: DAB2IP-knockdown of BCa cells (i.e., siDAB2IP) exhibit increased clonogenic survival in response to IR compared with control cells (i.e., siCON) expressing an endogenous level of DAB2IP. The mechanism in siDAB2IP cells could be explained by elevated ATM expression and activation, increased S phase cell distribution as well as faster DSB repair kinetics. 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one (KU55933) significantly sensitized siDAB2IP cells to IR due to inhibition of the phosphorylation of ATM and its downstream targets following IR and slower DSB repair kinetics. CONCLUSIONS: Loss of DAB2IP expression in BCa cells signifies their radioresistance. KU55933, which suppresses ATM phosphorylation upon irradiation, could be applied in the radiotherapy of BCa patients with a DAB2IP gene defect.

[The effect of Ixabepilone on NSCLC cells radio-sensitivity under quiescent or hypoxic condition].

OBJECTIVE: To evaluate the effect of Ixabepilone on quiescent or hypoxic cells response to ionizing radiation. METHODS: NCI-H460 and A549, two human NSCLC cell lines, were employed in this experiment. Quiescent cells (QC) or hypoxic cells (HC) were induced as mentioned previously and untreated cells as control. A colony forming assay was applied to compare cellular radio-sensitivity with or without Ixabepilone. Flow cytometry and Western blot analysis were used to detect cell cycle distribution and apoptosis. RESULTS: Along with an increased population of G1 cell (NCI-H46 P=0.001 3; A549 P=0.006), both HC and QC were exhibited radio-resistance compared to untreated cells (survival fraction: NCI-H460 P=0.003; A549 P=0.000 1). Moreover, it was found that Ixabepilone, which induced apoptosis in both IR-treated or untreated NSCLC cells, significantly enhanced cells death under hypoxia (decrease of survival fraction: NCI-H460 P=0.000 2; A549 P<0.01). CONCLUSION: The existence of quiescent or hypoxic cells in solid tumors poses a critical therapeutic problem since they were resistant to IR. Ixabepilone, which induces apoptosis in NSCLC, showed great radio-sensitization effect on hypoxic cells. Following work will focus on whether Ixabepilone could increase hypoxic tumor cells radio-sensitivity in vivo, which could provide useful data for the application of Ixabepilone in clinical practice.

Downregulation of DAB2IP results in cell proliferation and invasion and contributes to unfavorable outcomes in bladder cancer.

The DOC-2/DAB2 interactive protein (DAB2IP) is a member of the Ras GTPase-activating protein family. It has been shown to be often downregulated and a poor prognostic factor in several human malignancies. In this study, we analyzed the clinicopathological features and outcomes of DAB2IP expression in 135 patients with urothelial carcinoma of the bladder (UCB) treated by radical cystectomy plus bilateral lymph node dissection, and evaluated the effect of DAB2IP knockdown in vitro using the MTT method, colony formation assay, cell cycle assay, and cell migration and invasive assay. We found low expression of DAB2IP was significantly associated with high pathological stage (P = 0.002), high pathological grade (P = 0.02), tumor size more than 3 cm (P = 0.04), and presence of histological variants (P = 0.01). DAB2IP was an independent prognostic factor of disease recurrence (hazard ratio, 2.67; P = 0.034) and cancer-specific survival (hazard ratio, 2.79; P = 0.038). Knockdown of DAB2IP could promote cell proliferation, migration, and invasion. Downregulation of DAB2IP could activate the ERK and Akt pathways and was correlated with the expression of epithelial-mesenchymal transition markers, such as E-cadherin and vimentin. In conclusion, downregulation of DAB2IP is associated with features of biologically aggressive UCB and results in cell proliferation, migration, and invasion of bladder cancer. DAB2IP may serve as a promising biomarker in patients with UCB treated by radical cystectomy and bilateral lymph node dissection.

Regulation of hypoxia inducible factor-1α expression by the alteration of redox status in HepG2 cells.

Hypoxia inducible factor-1 (HIF-1) has been considered as a critical transcriptional factor in response to hypoxia. It can increase P-glycoprotein (P-Gp) thus generating the resistant effect to chemotherapy. At present, the mechanism regulating HIF-1α is still not fully clear in hypoxic tumor cells. Intracellular redox status is closely correlated with hypoxic micro-environment, so we investigate whether alterations in the cellular redox status lead to the changes of HIF-1α expression. HepG2 cells were exposed to Buthionine sulphoximine (BSO) for 12 h prior to hypoxia treatment. The level of HIF-1α expression was measured by Western blot and immunocytochemistry assays. Reduce glutathione (GSH) concentrations in hypoxic cells were determined using glutathione reductase/5,5'-dithiobis-(2-nitrob-enzoic acid) (DTNB) recycling assay. To further confirm the effect of intracellular redox status on HIF-1α expression, N-acetylcysteine (NAC) was added to culture cells for 8 h before the hypoxia treatment. The levels of multidrug resistance gene-1 (MDR-1) and erythropoietin (EPO) mRNA targeted by HIF-1α in hypoxic cells were further determined with RT-PCR, and then the expression of P-Gp protein was observed by Western blotting. The results showed that BSO pretreatment down-regulated HIF-1α and the effect was concentration-dependent, on the other hand, the increases of intracellular GSH contents by NAC could partly elevate the levels of HIF-1α expression. The levels of P-Gp (MDR-1) and EPO were concomitant with the trend of HIF-1α expression. Therefore, our data indicate that the changes of redox status in hypoxic cells may regulate HIF-1α expression and provide valuable information on tumor chemotherapy.

Epothilone B confers radiation dose enhancement in DAB2IP gene knock-down radioresistant prostate cancer cells.

PURPOSE: In metastatic prostate cancer, DOC-2/DAB2 interactive protein (DAB2IP) is often downregulated and has been reported as a possible prognostic marker to predict the risk of aggressive prostate cancer (PCa). Our preliminary results show that DAB2IP-deficient PCa cells are radioresistant. In this study, we investigated the anticancer drug Epothilone B (EpoB) for the modulation of radiosensitivity in DAB2IP-deficient human PCa cells. METHODS AND MATERIALS: We used a stable DAB2IP-knock down human PCa cell line, PC3 shDAB2IP, treated with EpoB, ionizing radiation (IR), or the combined treatment of EpoB and IR. The modulation of radiosensitivity was determined by surviving fraction, cell cycle distribution, apoptosis, and DNA double-strand break (DSB) repair. For in vivo studies, the PC3shDAB2IP xenograft model was used in athymic nude mice. RESULTS: Treatment with EpoB at IC(50) dose (33.3 nM) increased cellular radiosensitivity in the DAB2IP-deficient cell line with a dose enhancement ratio of 2.36. EpoB delayed the DSB repair kinetics after IR and augmented the induction of apoptosis in irradiated cells after G(2)/M arrest. Combined treatment of EpoB and radiation enhanced tumor growth delay with an enhancement factor of 1.2. CONCLUSIONS: We have demonstrated a significant radiation dose enhancement using EpoB in DAB2IP-deficient prostate cancer cells. This radiosensitization can be attributed to delayed DSB repair, prolonged G(2) block, and increased apoptosis in cells entering the cell cycle after G(2)/M arrest.

Downregulation of human DAB2IP gene expression in prostate cancer cells results in resistance to ionizing radiation.

DAB2IP (DOC-2/DAB2 interactive protein) is a member of the RAS-GTPase-activating protein family. It is often downregulated in metastatic prostate cancer and has been reported as a possible prognostic marker to predict the risk of aggressive prostate cancer. In this study, we furnish several lines of evidence indicating that metastatic human prostate cancer PC3 cells deficient in DAB2IP (shDAB2IP) exhibit increased clonogenic survival in response to ionizing radiation (IR) compared with control cells expressing an endogenous level of DAB2IP (shVector). Radioresistance was also observed in normal prostate cells that are deficient in DAB2IP. This enhanced resistance to IR in DAB2IP-deficient prostate cancer cells is primarily due to faster DNA double-strand break (DSB) repair kinetics. More than 90% of DSBs were repaired in shDAB2IP cells by 8 hours after 2 Gy radiation, whereas only 60% of DSB repair were completed in shVector cells at the same time. Second, upon irradiation, DAB2IP-deficient cells enforced a robust G(2)-M cell cycle checkpoint compared with control cells. Finally, shDAB2IP cells showed resistance to IR-induced apoptosis that could result from a striking decrease in the expression levels of proapoptotic proteins caspase-3, caspase-8, and caspase-9, and significantly higher levels of antiapoptotic proteins Bcl-2 and STAT3 than those in shVector cells. In summary, DAB2IP plays a significant role in prostate cell survival following IR exposure due to enhanced DSB repair, robust G(2)-M checkpoint control, and resistance to IR-induced apoptosis. Therefore, it is important to identify patients with dysregulated DAB2IP for (a) assessing prostate cancer risk and (b) alternative treatment regimens.

[Effect of constitutive androstane receptor on the cytotoxicity of mitomycin C and 5-(aziridin-1-yl)-3-hydroxymethyl-1-methylindole-4,7-dione].

This study is to evaluate the cytotoxicity of mitomycin C (MMC) and its analogue 5-(aziridin-1-yl)-3-hydroxymethyl-1-methylindole-4,7-dione (629) as well as the effect of transfection of constitutive androstane receptor (CAR) on their biological effects. HepG2 cells were transfected with the plasmids mCAR1/pCR3 mediated by liposome. Vector pCR3 was used as control. Transfected cells were screened by G418 resistance and limiting dilution. The expressions of plasmid mCAR1/pCR3 and CYP2B6 mRNA were detected by RT-PCR; Cytotoxicities of MMC and 629 in vitro were evaluated in g2car cells and HepG2 cells by MTT method under anaerobic and aerobic conditions. mRNA expression of CAR and CYP2B6 can not be detected in HepG2 cells and HepG2/pCR3 cells but can in g2car cells. It is shown that plasmid mCAR1/pCR3 was transfected into g2car cells successfully and target CYP2B6 was transactivated by CAR. To compare with aerobic and anaerobic, the cytotoxicities of MMC and 629 to HepG2 cells and g2car cells had significantly enhanced (P < 0.05), and transfect CAR gene can improve the cytotoxicity of MMC (P < 0.05), but not 629 (P > 0.05). Furthermore, CYP2B6 is one master enzyme for the metabolism of MMC and not 629. Transfection of CAR can increase expression of CYP2B6 mRNA in HepG2 cells, and can affect cytotoxicities of MMC and 629.

[Effect of inducible nitric oxide synthase on tumour cells sensitivity to mitomycin C analogue 629 in vitro].

AIM: To examine the effect of inducible nitric oxide synthase (iNOS) on tumour cells chemosensitivity to mitomycin C (MMC) analogue 5-aziridinyl-3-hydroxyl-1-methylindole-4,7-dione (629) in vitro, and elucidate the possible role of iNOS in the metabolism of 629. METHODS: Human sarcoma cells (HT1080) and its iNOS gene transfected clones (iNOS9, iNOS12) were exposed to 629 at concentrations of 1 nmol x L(-1) - 100 micromol x L(-1). 3-[4, 5-Dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (MTT) assay, agarose electrophoresis and flow cytometric analysis were used to determine cell sensitivity, deoxyribonucleic acid (DNA) damage and the change of cell cycle in above process, respectively. All experiments were performed both in air and under hypoxia parallelly. RESULTS: 629 was more toxic than MMC, and enhanced cytotoxicity under hypoxia, which resulted in cell necrosis. Sixteen hours after treated with 629, HT1080 cells and related iNOS-transfected clone cells were obviously blocked in G2/M phase. CONCLUSION: iNOS plays dual roles in 629 metabolism, enhancing or decreasing the cytoxicity of 629 depending on the intracellular oxygen pressure P(O2), which caused higher cytotoxicity to hypoxia cells of 629 with the increasing of iNOS activity.