Radiosensitivity Of Prostate Cancer Cells Research Articles

Effects of p300 inhibition on prostate cancer cell growth

Background: Radiation is a central component of many anti-cancer therapy regimens, utilized for more than half of all cancer patients. Despite advances in the delivery and success of radiation treatment, many individuals express, or will develop, radioresistance, a major contributor to treatment failure and malignant progression. While the etiology of radioresistance is complex, it may be mediated in part by expression of p300, a histone acetyltransferase known to facilitate DNA repair. We hypothesized that inhibition of p300 would increase radiosensitivity of prostate cancer cells in vitro. Methods: Human prostate adenocarcinoma (PC-3) cells were grown in tissue culture flasks incubated at 37° C with 5% CO2. Cells were randomized to receive 1 μM CCS 1477, a selective inhibitor of p300, or vehicle for 1 hour prior to a single treatment of 0 Gy (NR+ or NR-, respectively) or 2 Gy radiation (R+ or R-, respectively). Four hours after radiation, cells were seeded into 100 mm diameter tissue culture dishes and incubated for 10 days. Cells were then stained, and colonies > 50 cells were counted to determine clonogenic survival. Cell survival was corrected for plating effciency and group comparisons were made using a two-way ANOVA. Results: Results were determined from the average of 3 separate experiments. Compared to non-radiated controls (NR-), survival of R- was reduced to 47%, while survival of R+ was reduced to 32%. However, there was no significant difference between % survival of R- and R+. Conclusions: In vitro, 1 μM exposure to CCS 1477 does not reduce clonogenic survival in PC-3 cells subjected to 2 Gy radiation. The duration of exposure and concentration of CCS 1477 may not be adequate to elicit significant improvements in radiosensitivity of PC-3 cells to 2 Gy radiation. Future experiments will explore the effects of varying durations of exposure and concentrations of CCS 1477. Additionally, it remains to be determined whether CCS 1477 improves radiosensitivity in other cancer cell lines (e.g., breast cancer). Johnson Cancer Research Center. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Disruption of mitochondrial function augments the radiosensitivity of prostate cancer cell lines.

Ionizing radiation is one of the most widely used therapeutic methods in the treatment of prostate cancer, but the problem is developing radioresistance of the tumour. There is evidence that metabolic reprogramming in cancer is one of the major contributors to radioresistance and mitochondria play a crucial role in this process. The aim of the study was to assess the influence of oxidative phosphorylation uncoupling to radiosensitivity of prostate cancer cells differing in metabolic phenotype. LNCaP, PC-3 and DU-145 cells were exposed to X-rays and simultaneously treated with 2,4-dinitrophenol (2,4-DNP). The radiosensitive of cell lines was determined by cell clonogenic assay and cell cycle analysis. The cytotoxic effect was evaluated with MTT and CVS (Crystal violet staining) assay, apoptosis detection and cell cycle analysis. The phenotype of the cells was determined by glucose uptake and lactate release, ATP level measurement as well as basal reactive oxygen species level and mRNA expression of genes related to oxidative stress defence. The synergistic effect of 2,4-dinitrophenol and X-ray was observed only in the case of the LNCaP cell line. Phenotypic analysis indicates that this may be due to the highest dependence of these cells on oxidative phosphorylation and sensitivity to disruption of their redox status.

Radiosensitization of PC3 Prostate Cancer Cells by 5-Thiocyanato-2'-deoxyuridine.

Simple SummaryRadiation therapy is one of the main treatments for cancer. However, the success of treatment by radiation therapy is largely dependent on tumor radiosensitivity. To improve therapeutic outcomes, radiation therapy should be combined with the use of a radiosensitizer which enables irradiation at lower doses with higher efficacies. 5-Thiocyanato-2′-deoxyuridine has been reported as a potential radiosensitizer of DNA damage based on advanced radiation chemical studies. In this paper, for the first time, we demonstrate the radiosensitizing properties of this modified nucleoside at the cellular level. The tested analogue increases the sensitivity of prostate cancer cells to ionizing radiation which is, at least partially, related to an increase in the number of DNA double-strand breaks and cell cycle regulation.Purpose: The radiosensitizing properties of uracil analogs modified in the C5 position are very interesting in the context of their effectiveness and safety in radiation therapy. Recently, radiation chemical studies have confirmed that 5-thiocyanato-2′-deoxyuridine (SCNdU) undergoes dissociation induced by an excess electron attachment and established this nucleoside as a potential radiosensitizer. In this paper, we verify the sensitizing properties of SCNdU at the cellular level and prove that it can effectively enhance ionizing radiation-induced cellular death. Methods and Materials: Prostate cancer cells were treated with SCNdU and irradiated with X rays. The cytotoxicity of SCNdU was determined by MTT test. Cell proliferation was assessed using a clonogenic assay. Cell cycle analyses, DNA damage, and cell death analyses were performed by flow cytometry. Results: SCNdU treatment significantly suppressed the proliferation and increased the radiosensitivity of prostate cancer cells. The radiosensitizing effect expressed by the dose enhancement factor is equal to 1.69. Simultaneous exposure of cells to SCNdU and radiation causes an increase in the fraction of the most radiosensitive G2/M phase, enhancement of the histone H2A.X phosphorylation level, and apoptosis induction. Finally, SCNdU turned out to be marginally cytotoxic in the absence of ionizing radiation. Conclusions: Our findings indicate that SCNdU treatment enhances the radiosensitivity of prostate cancer cells in a manner associated with the cell cycle regulation, double strand formation, and a slight induction of apoptosis.

Knockdown of MAGE-A6 enhanced the irradiation sensitivity of non-small cell lung cancer cells by activating the AMPK pathway.

Non-small cell lung cancer is a common respiratory tumor. The mortality rate of lung cancer patients has continued to rise in recent years. Several studies revealed that the expression of melanoma antigen 6 (MAGE-A6) promoted the development of multiple types of cancer. In addition, the suppression of AMPK pathway could restrict the radiosensitization of prostate cancer cells. Inhibition of MAGE-A6 activated the AMPK pathway in colorectal cancer cells. However, whether the MAGE-A6 could regulate the radiosensitivity of non-small cell lung cancer cells by regulating of the AMPK pathway is unclear. In this study, we established the MAGE-A6 knockdown in A549 and H1299 cells. Next, the apoptosis and proliferation of these cells were detected by the flow cytometry analysis and colony formation assay after the irradiation, respectively. Then, the expression of p-AMPKα1 and p-S6K1 in these cells was explored by the western blotting. After that, we inhibited the expression of AMPKα1 in MAGE-A6 knockdown cells. The proliferation and apoptosis of these cells were detected with colony formation assay and flow cytometry analysis. Finally, the tumor formation of these cells was detected in nude mice. Our results showed that inhibition of MAGE-A6 suppressed the proliferation and aggravated the apoptosis of A549 and H1299 cells after the irradiation. Knockdown of MAGE-A6 activated the expression of p-AMPKα1 and repressed the expression of p-S6K1 in these cells. Suppression of AMPKα1 in MAGE-A6 knockdown cells abolished these effects. Knockdown of MAGE-A6 also enhanced the radiosensitivity of these cells in vivo. These results suggested that inhibition of MAGE-A6 promoted the radiosensitivity of non-small cell lung cancer cells by activating AMPK pathway. Therefore, MAGE-6 has the potential to be explored as the therapeutic target for the treatment of non-small cell lung cancer in clinical.

STAT3 as a target for sensitizing prostate cancer cells to irradiation.

Radioresistance of prostate cancer (PCa) is a major factor leading to local failure of radiotherapy. STAT3 is an oncogenic protein that was recently found to be activated in PCa tumors. This study aimed to investigate the radiosensitization effect of targeting STAT3 in PCa tumors. Here, the radiosensitization effect of STAT3 blockade was investigated by clonogenic assay, flow cytometry and western blot analysis in human PCa cells in vitro and in vivo. We demonstrated that STAT3 blockade with a STAT3 inhibitor or siRNA increased the radiosensitivity of PCa cells and that radiation together with STAT3 blockade induced more apoptosis and double-strand breaks (DSBs) than radiation alone in LNCaP cells. In addition, radiation induced STAT3 activation and survivin expression in PCa cells, which was inhibited by STAT3 blockade. Transfection with survivin cDNA attenuated the radiosensitization effect of STAT3 blockade. These effects were further confirmed by in vivo studies, which showed that the STAT3 inhibitor enhanced the treatment efficacy of radiation on LNCaP xenografts with decreased STAT3 activation and survivin expression.These findings suggest that STAT3 blockade radiosensitizes PCa cells through regulation of survivin. Thus, our study has revealed STAT3 as a potential sensitizer for irradiation in PCa cells. Its clinical application as an adjuvant in radiotherapy of PCa should be explored in the future.

Impact of PSMA Targeted Gold Nanoparticles on Prostate Cancer Radiation Bystander Effect

Gold nanoparticles (GNPs) are effective radiosensitizers due to their ability to enhance ionizing radiation absorptance and deposit elevated dose in their vicinity. Radiation induced bystander effect (RIBE) expands radiation effects to non-targeted cells close to radiation-targeted cells, further improving radiation outcome. In this study, we investigated the impact of PSMA-targeted GNPs on RIBE. We hypothesize that the PSMA-targeted GNPs would intensify radiation effects by augmenting RIBE consequence as well as enhancing sensitivity of bystander cells to RIBE.Anti-prostate-specific membrane antigen (PSMA) antibodies were conjugated onto PE Gylated GNPs through EDC/NHS chemistry. UV-Vis spectrophotometry and real time cell analysis were employed to evaluate the cellular uptake and biocompatibility, respectively, when LNCaP prostate cancer cells were treated with PSMA-targeted GNPs at varying concentrations. Then GNPs with biocompatible dosage were used for cancer cell treatments. The media transfer technique was performed to induce the RIBE effects on bystander cells. First, LNCaP cells pretreated with phosphate buffered saline (PBS) or GNPs for 24hrs were irradiated with 160kvP X-rays at varying doses (0∼8 Gy). The culture media from irradiated cells were collected and filtered (sterile 0.2µm polyethersulfone) to acquire PBS conditioned media (PBS-CM) and GNP conditioned media (GNP-CM). Then, the collected PBS-/GNP-CMs were transferred to non-irradiated LNCaP cells with/without GNPs for 24hrs to investigate RIBE effect. The final GNP-induced RIBE was evaluated via MTT cellular viability assay, clonogenic assay, γ-H2AX assay, as well as reactive oxygen species (ROS) assessment through 2',7'-dichlorofluorescein diacetate staining.PSMA-targeted GNPs showed minimal cytotoxicity in LNCaP cells up to 1mg/mL concentrations. Cellar uptake analysis demonstrated that GNPs were bound to cells within 30 mins, with maximum uptake at 250µg/mL. Compared to 2 Gy PBS-CM, 8 Gy PBS-CM demonstrated much higher RIBE response on the non-irradiated cells, showing the dose dependent RIBE in LNCaP cells. Compared to PBS-CM, GNP-CM exhibited much higher cell death in MTT assay, as well as demonstrated RIBE enhancement in γ-H2AX (133%) and clonogenic assays (120%) analysis. Moreover, GNP-CM showed elevated ROS expression (125%) than PBS-CM. In the presence of PBS-CM, the non-irradiated cells with GNPs showed increased cell death compared to that without GNPs, indicating that GNP would enhance the sensitivity of bystander cells to RIBE. This sensitivity enhancement ratio was also validated in γ-H2AX (120%) and clonogenic assays (120%).PSMA directed GNPs were associated with physical dose enhancement along with the enhanced RIBE, playing a critical role in increasing the overall radiosensitivity of prostate cancer cells. Our results also showed that PSMA-targeted GNPs enhanced the sensitivity of bystander cells to RIBE.

Effects of Heat Therapy on Prostate Cancer Cells Subjected to 4 Gy Radiation

Background Despite many advancements in treatment options, radiation therapy remains a common treatment for individuals diagnosed with prostate cancer. However, radiation resistance presents a significant challenge for many patients. Due to its numerous effects on cancer cell growth and survival, heat therapy has been studied as a potential mechanism to improve radiation efficacy. However, the modality of heat therapy varies widely between studies, and the majority of heat therapy research relies on non-physiological temperatures (>43º C). We hypothesized that mild temperature hyperthermia would improve the efficacy of radiation of prostate cancer cells in vitro. Methods Clonogenic survival was assessed using human prostate cancer (PC-3) cells in vitro. Cells were grown in tissue culture flasks maintained in a 37º C incubator with 5% CO2 and randomized into 3 groups: normothermic (NT; n=8), acute hyperthermia (HTA; n=8), and chronic hyperthermia (HTC; n=8). NT cells were maintained in a normal incubator until radiation. For heat treatments, HTA cells were heated to 41.0º C in a separate incubator for 1 h immediately prior to radiation. HTC cells were heated to 41.0º C in a separate incubator for 1 h each session, for a total of 3 heat treatments, each separated by 48 h, with the final heat treatment occurring 24 h prior to radiation. Immediately following a single dose of 4 Gy radiation, each group of cells were counted and plated into 60 mm culture dishes at densities of both 500 cells/plate and 1000 cells/plate, with 5 replicates plated per density. Following 8 days of incubation, cells were fixed and stained and colonies of greater than 50 cells were counted. Results The survival fractions were determined from the average of the two plating densities. Clonogenic cell survival was significantly reduced in HTA vs. NT (11.6% ± 2% vs. 19.3% ± 1%, p<0.05) and in HTC vs. NT (8.3% ± 2% vs. 19.3% ± 1%, p<0.05). Cell survival between HTA and HTC was not different (p>0.05). Conclusions These results suggest that both acute and chronic mild hyperthermia improve the efficacy of 4 Gy radiation in PC-3 cells. While exact mechanisms are yet to be elucidated, it is thought that hyperthermia at lower temperature induces DNA damage, inhibits DNA damage repair, or alters cell cycle progression, which individually or in combination with one another, may potentiate the effects of radiation. Mild temperature hyperthermia prior to radiation may be an effective neo-adjuvant to increase the radiosensitivity of prostate cancer cells.

MiR-541-3p enhances the radiosensitivity of prostate cancer cells by inhibiting HSP27 expression and downregulating \u03b2-catenin

Heat shock protein 27 (HSP27), a regulator of cell survival, can enhance the resistance of cancer cells to radiotherapy. As microRNA-541-3p (miR-541-3p) was recently predicted to be a putative upstream modulator of HSP27, the present study was designed to investigate the function and mechanism underlying how miR-541-3p modulates the radiosensitivity of prostate cancer (PCa) cells by regulating HSP27. Through quantitative PCR, miR-541-3p was determined to be poorly expressed in PCa tissues relative to normal controls, whereas its expression was enhanced after radiotherapy. Consistently, miR-541-3p expression levels in PCa cells were elevated after radiation. Cell viability and proliferation and apoptosis under radiation were subsequently evaluated in response to loss-of-function of miR-541-3p. It was found that inhibition of miR-541-3p facilitated the viability and proliferation of PCa cells and promoted their apoptosis post radiation, hence reducing the radiosensitivity of LNCaP cells. Dual-luciferase reporter assay identified that miR-541-3p negatively regulated the HSP27 mRNA expression by targeting its 3′-UTR. Meanwhile, miR-541-3p overexpression inhibited the β-catenin expression by targeting HSP27. Furthermore, HSP27 or β-catenin overexpression was noted to significantly reverse the miR-541-3p-mediated changes in the biological functions of PCa cells post radiation, suggesting that HSP27-dependent activation of β-catenin might be the mechanism responsible for the promotive effect of miR-541-3p on radiosensitivity. Collectively, this study suggests that miR-541-3p specifically inhibits the HSP27 expression and downregulates β-catenin, thereby enhancing the radiosensitivity of PCa cells. Our findings highlight the underlying mechanism of the miR-541-3p/HSP27/Wnt/β-catenin axis regarding radiotherapy for PCa.

Circ_0062020 Knockdown Strengthens the Radiosensitivity of Prostate Cancer Cells.

BackgroundProstate cancer (PCa) is a major contributor to reduce the life quality of males. Circular RNAs were frequently reported to be associated with cancers. In the case of radiotherapy to PCa, the role of circ_0062020 was still inconclusive, which was further explored in this study.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the expression of circ_0062020, miR-615-5p and thyroid hormone receptor interactor 13 (TRIP13) in PCa tissues and cells, as well as in normal tissues and cell. Meanwhile, the proliferation of PCa cells was evaluated by clone formation assay and cell counting kit 8 (CCK8) assay. Moreover, the metastasis of PCa cells was assessed by transwell and wound healing assays. Furthermore, the apoptosis of PCa cells was determined by flow cytometry assay. Besides, dual-luciferase reporter system was applied to verify the correlation between miR-615-5p and circ_0062020 or TRIP13, which was predicted by online tool CircRNA interactome or TargetScan. In addition, the protein expression of TRIP13 was measured by Western blot in PCa tissues and cells and normal tissues and cells. Finally, xenograft tumor assay was performed to further confirming the function of circ_0062020 in PCa in vivo.ResultsCirc_0062020 and TRIP13 were upregulated, while miR-615-5p was downregulated in PCa tissues and cells. Circ_0062020 knockdown or miR-615-5p overexpression inhibited the proliferation and metastasis, and promoted apoptosis, which could be reversed by miR-615-5p inhibitor or pc-TRIP13 in ionizing radiation (IR)-treated PCa cells. As expected, circ_0062020 sponged miR-615-5p to regulate TRIP13 expression in PCa cells. Circ_0062020 knockdown also suppressed PCa tumor growth in vivo.ConclusionCirc_0062020 suppressed the radiosensitivity by miR-615-5p/TRIP13 axis in PCa cells, which might provide insights into the radiotherapy for PCa.

Tumor cell intrinsic androgen biosynthesis by 3β-hydroxy steroid dehydrogenase (HSD3B1) to modulate radiosensitivity in prostate cancer cells.

349 Background: Resistance to ADT is associated with a gain of function mutation in the 3β-HSD enzyme, which catalyzes extragonadal/intratumoral DHT synthesis. As androgen signaling is known to upregulate the DNA damage response (DDR), we investigated whether HSD3B1 genotype modulates DDR and radiosensitivity in PCa. Methods: We stably knocked down HSD3B1 in LNCaP, C42 and VCaP cell lines (which carry the protein stabilizing variant allele) and overexpressed the variant HSD3B1 allele in LAPC4 (harbors a WT allele which readily undergoes degradation). We examined the proliferative and clonogenic capacity of these cells in presence and absence of substrate, DHEA, followed by treatment with IR (400-800 cGy, single fraction). We studied DNA DSB formation and resolution kinetics using γH2AX foci formation in response to radiation. We also measured changes in mRNA expression of DDR response genes pre- and post-radiation. Results: Control shRNA transduced cell lines had increased cell proliferation (p&lt;0.001) and clonogenic survival (2 logs at 800cGy single fraction radiation, p&lt;0.001) in the presence of DHEA compared to HSD3B1 knockdown cells. Variant HSD3B1 cell lines were more radioresistant and exhibited more efficient γH2AX foci resolution at 24 hrs (p &lt;0.05) in a DHEA dependent manner. We observe increased mRNA expression of DDR genes from specific repair networks including non-homologous end joining (PRKDC, XRCC4, XRCC5) and homologous recombination (RAD51, RAD54) in variant HSD3B1 cells. Transcriptional induction of DDR genes following radiation in presence of DHEA was significantly more pronounced in HSD3B1 variant cells, suggesting a more permissive chromatin context. Conclusions: Increased intracellular 3β-HSD drives transcription of NHEJ and HR genes, more rapid resolution of γH2AX foci, and radioresistance in prostate cancer. This work has therapeutic implications related to strategies for combined radiation and androgen directed therapy in localized and metastatic prostate cancer. Prospective validation of treatment strategies combining blockade of adrenal steroid precursor synthesis, ADT, and XRT in high risk disease is warranted.

Knockdown of FBP1 enhances radiosensitivity in prostate cancer cells by activating autophagy.

For patients with clinically early-stage localized prostate cancer, radiotherapy is another treatment that can achieve radical treatment in addition to radical prostatectomy. Despite this, there are still a large number of patients with prostate cancer who have a biochemical recurrence after undergoing radiotherapy, or even clinical recurrence, leading to treatment failure. Although the expression of the fructose-1,6-bisphosphatase 1 (FBP1) gene has been found to be absent in various tumors and is associated with a poor prognosis in tumor patients. However, the expression and role of FBP1 in prostate cancer are not clear. The purpose of this study was to investigate the role and mechanism of FBP1 in the radiotherapy resistance of prostate cancer. By analyzing the microarray data of prostate cancer radiotherapy resistant cells and parental cells (GSE53902), we found that FBP1 expression in DU145 radiotherapy resistant cells was significantly higher than in the DU145 parental cells. In addition, we searched for the expression of FBP1 in 492 prostate cancer samples from TCGA and found that its expression in prostate cancer was significantly higher than that in normal tissues. Knockdown of FBP1 expression significantly inhibited the proliferation of prostate cancer cells, promoted DNA damage-mediated apoptosis, and enhanced the sensitivity of prostate cancer cells to radiotherapy. Further mechanism analysis revealed that FBP1 knockdown could activate autophagy mediated by the AMPK-mTOR signaling pathway, while inhibition of the AMPK-mTOR signaling pathway could reverse FBP1 knockdown-mediated autophagy and apoptosis, as well as radiosensitization. In conclusion, this study clarified that FBP1 is an oncogene in prostate cancer, and the main mechanism for knockdown of FBP1 to increase radiosensitivity is to enhance autophagy mediated by the AMPK-mTOR signaling pathway. Therefore, FBP1 may be a potential target for enhancing prostate cancer radiotherapy.

Fatty Acid Inhibition Sensitizes Androgen-Dependent and -Independent Prostate Cancer to Radiotherapy via FASN/NF-\u03baB Pathway

Elevated fatty acid synthase (FASN) has been reported in both androgen-dependent and -independent prostate cancers. Conventional treatment for prostate cancer is radiotherapy (RT); however, the following radiation-induced radioresistance often causes treatment failure. Upstream proteins of FASN such as Akt and NF-κB are found increased in the radioresistant prostate cancer cells. Nevertheless, whether inhibition of FASN could improve RT outcomes and reverse radiosensitivity of prostate cancer cells is still unknown. Here, we hypothesised that orlistat, a FASN inhibitor, could improve RT outcomes in prostate cancer. Orlistat treatment significantly reduced the S phase population in both androgen-dependent and -independent prostate cancer cells. Combination of orlistat and RT significantly decreased NF-κB activity and related downstream proteins in both prostate cancer cells. Combination effect of orlistat and RT was further investigated in both LNCaP and PC3 tumour-bearing mice. Combination treatment showed the best tumour inhibition compared to that of orlistat alone or RT alone. These results suggest that prostate cancer treated by conventional RT could be improved by orlistat via inhibition of FASN.

Radiosensitization and Micronucleus Formation are Induced by Centrosome Clustering Inhibition with Griseofulvin in Prostate Cancer Cell Lines

Ionizing radiation (IR) leads to micronucleus-dependent immunogenic signaling through cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), and type I interferon. Inhibition of supernumerary centrosome clustering in tumor cells with griseofulvin can increase micronucleus formation and mitotic cell death but has not been studied in prostate cancer. We hypothesize that co-administration of IR and centrosome clustering inhibition (CCi) will radiosensitize prostate cancer cells and increase cGAS signaling, a potential avenue for enhancing the interplay between radiotherapy and immunotherapy. Murine Myc-CaP and human PC3 and 22rv1 prostate cancer cells were grown in culture using standard techniques. CCi was achieved with griseofulvin. Clonogenic survival was assessed across a range of griseofulvin (3-30 μM) and IR (1-12 Gy) doses, both as single interventions and combination treatments. Micronuclei (MN) were visualized and quantified by DAPI staining. Bipolar and multipolar spindles were identified by immunofluorescence against alpha-tubulin and pericentrin. In untreated Myc-CaP cells, MN were identified in 2.8% of cells. Griseofulvin treatment resulted in dose-dependent increases in MN, seen in 15% of cells after 30 μM treatment. IR also resulted in dose-dependent MN formation, with 4 Gy inducing MN in 24% of cells. Similar effects were observed in PC3 and 22rv1 cells, with 30 μM griseofulvin inducing MN in 5.2% and 5.8% of cells, while 9 Gy IR induced MN in 7.4% and 11.7%. After treatment 30 μM griseofulvin, multipolar spindles were identified in 31.3% of Myc-Cap, 10% of PC3, and 18.5% of 22rv1 cells, as compared to 4.6%, 4%, and 0% in untreated cells. Pretreatment of Myc-CaP cells with 30 μM griseofulvin prior to 3 Gy IR resulted in supra-additive decreases in clonogenic survival with a Chou-Talalay combination index (CI) of 0.69. In PC3 and 22rv1 cells, 30 μM griseofulvin enhanced 1 Gy IR with CI of 0.25 and 0.31, respectively. Griseofulvin and IR induce dose-dependent micronucleus formation in murine and human prostate cancer cell lines, and griseofulvin also promotes multipolar mitosis. Treatment with griseofulvin prior to IR decreases clonogenic survival across tested cell lines. These data suggest that combination treatment with griseofulvin and IR may improve local responses to radiotherapy and, through micronucleus-dependent immunogenic signaling, lead to more effective abscopal response generation in metastatic prostate cancer. Immediate future directions include investigation of cGAS/STING/interferon signaling axis and evaluation in an immune competent syngeneic mouse model with concurrent immune checkpoint inhibition.

αvβ3 Integrin Mediates Radioresistance of Prostate Cancer Cells through Regulation of Survivin.

The αvβ3 integrin is involved in various physiologic and pathologic processes such as wound healing, angiogenesis, tumor growth, and metastasis. The impact of αvβ3 integrin on the radiosensitivity of prostate cancer cells and the molecular mechanism controlling cell survival in response to ionizing radiation (IR) was investigated. Both LNCaP cells stably transfected with αvβ3 integrin and PC-3 cells that contain endogenous β3 integrin were used. This study demonstrated that αvβ3 integrin increases survival of αvβ3-LNCaP cells upon IR while small hairpin RNA (shRNA)-mediated knockdown of αvβ3 integrin in PC-3 cells sensitizes to radiation. Expression of αvβ3 integrin in LNCaP cells also enhances anchorage-independent cell growth while knockdown of αvβ3 integrin in PC-3 cells inhibits anchorage-independent cell growth. The αvβ3 antagonist, cRGD, significantly increases radiosensitivity in both αvβ3-LNCaP and PC-3 cells. Moreover, αvβ3 integrin prevents radiation-induced downregulation of survivin. Inhibition of survivin expression by siRNA or shRNA enhances IR-induced inhibition of anchorage-independent cell growth. Overexpression of wild-type survivin in PC-3 cells treated with αvβ3 integrin shRNA increases survival of cells upon IR. These findings reveal that αvβ3 integrin promotes radioresistance and regulates survivin levels in response to IR. IMPLICATIONS: Future translational research on targeting αvβ3 integrin and survivin may reveal novel approaches as an adjunct to radiotherapy for patients with prostate cancer.

128 - RelB-BLNK axis: a novel determinant of cell fate

Prostate cancer (PCa), the second leading cause of male cancer deaths in the United States, is most frequently treated with radiation therapy (RT). We screened 786 FDA-approved compounds to identify drugs that can radiosensitize PCa cells while radioprotecting non-cancer prostate epithelial cells and identified Betamethasone (BET) as a lead compound with these properties. BET increases ROS levels in PCa cells (high oxidized redox state) but not in normal prostate epithelial cells (low oxidized redox state). Remarkably, BET has an opposite effect on RelB, a non-canonical redox-sensitive NF-κB pathway. BET suppresses the expression of RelB and RelB target genes including MnSOD and survivin in PCa cells, while it induces expression of RelB and MnSOD in non-cancer cells. The differential effects of BET are, in part, attributable to the intrinsic differences in the cellular redox states of normal versus PCa cells. Intriguingly, RNA seq and ChIP assays identify B-cell linker protein (BLNK) as a novel RelB transcription regulator that is differentially regulated by BET in normal and cancer cells. Proximity-ligation assays and knockdown of BLNK confirm that RelB-BLNK possibly interact in the nuclear compartment. BET/RT treatments that target disruptions of RelB and BLNK expressions reverse the survival of PCa cells but not of normal prostate epithelial cells. Our study is the first to establish that the RelB-BLNK mediated NF-κB non-canonical pathway is a novel mechanism to regulate the differential responses to radiation therapy of normal prostate epithelial and prostate cancer cells. The results of this study fill in a critical gap in scientific knowledge of how the differences in redox conditions of normal and tumor cells trigger differential responses to pro-oxidants. They will facilitate the translation of therapeutic approaches that use prooxidants to push cancer cells into oxidative stress overload but stimulate adaptive responses in normal cells to selectively enhance RT efficacy.

Hydroxyflutamide Induces Androgen Receptor-Mediated DNA Damage and Radiosensitizes Prostate Cancer Cells While Preventing Induction of Pro-Growth Programs

Hydroxyflutamide Induces Androgen Receptor-Mediated DNA Damage and Radiosensitizes Prostate Cancer Cells While Preventing Induction of Pro-Growth Programs

Abstract A032: miR-205 reconstitution sensitizes prostate cancer cells and xenografts to radiotherapy, through PKCε suppression

Abstract Radiotherapy is a standard treatment for organ-confined prostate cancer (PCa). Although treatment technical optimization has greatly improved local tumor control, a significant percentage of patients still undergo recurrence. The onset of radioresistance is a complex phenomenon involving several genetic and epigenetic abnormalities, including the deregulation of microRNAs (miRNAs), endogenous small noncoding RNA molecules that negatively regulate gene expression. In this study, we investigated the ability of miR-205 to radiosensitize PCa cells. The possible relevance of the miRNA in PCa radiation response is based on our previous observation that miR-205 reconstitution in PCa cells i) exerts a tumor-suppressive effect in human PCa cells by counteracting epithelial-mesenchymal transition (EMT) and reducing cell migration/invasion, at least in part through the downregulation of its target PKCε, shown to play a role also in the nuclear translocation of EGFR, and ii) counteracts CAF-induced EMT, thus impairing enhancement of tumor cell invasion, acquisition of stem cell traits, tumorigenicity, and metastatic dissemination. The biologic role of miR-205 was elucidated through reconstitution in DU145 PCa cells by using a miRNA mimic. Radiation response in miRNA-reconstituted and control cells was assessed by the clonogenic assay. Transfected cells were exposed to 2-8 Gy delivered as a single dose using the 137Cs γ-irradiator IBL-437. Xenografts generated in mice following s.c. transplantation of DU145 cells stably expressing miR-205 received 5 Gy single-dose irradiation using a micro-CT/microirradiator (225Cx, Precision X-ray). We found that transient or stable reconstitution of miR-205 in DU145 cells consistently increased in vitro radiosensitivity. Among miR-205 direct targets, we were able to attribute a functional role in the radiosensitizing effect to PKCε. Indeed, siRNA-mediated silencing of PKCε in DU145 cells fully reproduced the enhanced radiation response induced by the miRNA. The effect was mediated, at least in part, by a decreased radiation-induced EMT and a reduced Non-Homologous End-Joining-based repair, as a consequence of the inhibited EGFR nuclear translocation. Based on our previous evidence indicating the ability of miR-205 to improve the activity of DNA damaging drugs through the downregulation of the lysosome-associated protein LAMP3 and autophagy impairment, we assessed the effect of LAMP3 knockdown on DU145 cell radiosensitivity and observed an enhanced radiation response, though at a lower extent compared to that induced by miR-205 reconstitution. The miRNA-mediated in vitro radiosensitizing effect was also appreciable in vivo. Indeed, xenografts generated by DU145 cells stably expressing the miRNA showed a significantly enhanced tumor growth inhibition following 5 Gy single-dose irradiation, as compared to control xenografts. Moreover, miR-205 reconstitution significantly increased the time for DU145 xenografts to reach 1,000 mm3 tumor burden. Consistent with in vitro findings, immunohistochemistry data obtained in tumor sections from miR-205-reconstituted xenografts revealed decreased PKCε expression. Overall, our results suggest miR-205 as a possible relevant determinant of ionizing radiation response, supporting the clinical interest in developing a novel therapeutic approach based on miR-205 reconstitution to increase PCa sensitivity to radiotherapy. Citation Format: Paolo Gandellini, Rihan El Bezawy, Marco Folini, Denis Cominetti, Stella Tinelli, Tiziana Rancati, Riccardo Valdagni, Nadia Zaffaroni. miR-205 reconstitution sensitizes prostate cancer cells and xenografts to radiotherapy, through PKCε suppression [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A032.

HZ08 suppresses RelB-activated MnSOD expression and enhances Radiosensitivity of prostate Cancer cells

BackgroundThe development of radioresistance is one of main causes for therapeutic failure of prostate cancer (PCa). The present study aims to investigate the function and the related mechanism by which HZ08 sensitizes radiotherapeutic efficiency to treat aggressive PCa cells.MethodsPCa cells were pretreated with HZ08 (6,7-dimethoxy-1-(3,4-dimethoxy) benzyl-2-(N-n-octyl-N′-cyano) guanyl-1,2,3,4-tetrahydroisoquinoline) and followed by ionizing radiation (IR) treatment. Cytotoxicity in the treated cells was analyzed to assess the radiosensitization capacity of HZ08 by flow cytometry, MTT and colony survival assays. The cellular levels of reactive oxygen species (ROS) and oxygen consumption rates (OCR) were measured using specific ROS detection probes and a Seahorse XF96 Analyzer, respectively. RelB binding to the NF-κB intronic enhancer region of the human SOD2 gene was determined using a ChIP assay. The levels of phosphorylation of PI3K, Akt and IKKα were quantified and further confirmed using a PI3K inhibitor. Finally, the synergistic effect of HZ08 on radiosensitization of PCa cells was validated using a mouse xenograft tumor model.ResultsHZ08 enhanced radiosensitivity of PCa cells through increasing ROS and declining mitochondrial respiration due to suppression of mitochondrial antioxidant enzyme MnSOD. Mechanistically, HZ08 appeared to inhibit PI3K/Akt/IKKα signaling axis, resulting in transcriptional repression of MnSOD expression by preventing RelB nuclear translocation.ConclusionsHZ08 can serve as a useful radiosensitizing agent to improve radiotherapy for treating aggressive PCa cells with high level of constitutive RelB. The present study suggests a promising approach for enhancing radiotherapeutic efficiency to treat advanced PCa by inhibiting antioxidant defense function.

Exendin-4 enhances radiation response of prostate cancer.

Exendin-4, one of the most widely used antidiabetic drugs, has recently been reported to have potential antitumor effects in cancers. Prostate cancer (PC) is one of the most common cancers in male patients with type 2 diabetes mellitus, and radiotherapy plays a vital role in the therapy of PC. Whether exendin-4 has the potential to enhance PC response to ionizing radiation (IR) remains unknown. We aimed to explore whether exendin-4 radiosensitizes PC cells. GLP-1 receptor (GLP-1R) expression in PC tissue samples and cell lines were analyzed, Human prostate cancer cells (PC3 and LNCap) were treated with IR and exendin-4, and subjected to proliferation, clone formation, cell cycle, immunoblotting, and immunohistochemical analysis. An in situ prostate tumor of animal model was established. We found that GLP-1R was expressed in human PC tissues and cell lines. 1-100 nM exendin-4 promoted the anti-proliferation effects of IR in vitro and in vivo, and enhanced radiation-induced G2/M cycle arrest in PC cells in a dose-dependent manner. Furthermore, Ex-4 increased AMPK phosphorylation, decrease the levels of p-mTOR, cyclin B, and p34cdc2 . Our study suggested exendin-4 radiosensitizes PC cells via activation of AMPK A and subsequent inhibition of p-mTOR, cyclin B, and p34cdc2 activation.

Disruption of MEK/ERK/c-Myc signaling radiosensitizes prostate cancer cells in vitro and in vivo.

Prostate cancer (PCa) cell radioresistance causes the failure of radiation therapy (RT) in localized or locally advanced disease. The aberrant accumulation of c-Myc oncoprotein, known to promote PCa onset and progression, may be due to the control of gene transcription and/or MEK/ERK-regulated protein stabilization. Here, we investigated the role of MEK/ERK signaling in PCa. LnCAP, 22Rv1, DU145, and PC3 PCa cell lines were used in in vitro and in vivo experiments. U0126, trametinib MEK/ERK inhibitors, and c-Myc shRNAs were used. Radiation was delivered using an x-6 MV photon linear accelerator. U0126 in vivo activity alone or in combination with irradiation was determined in murine xenografts. Inhibition of MEK/ERK signaling down-regulated c-Myc protein in PCa cell lines to varying extents by affecting expression of RNA and protein, which in turn determined radiosensitization in in vitro and in vivo xenograft models of PCa cells. The crucial role played by c-Myc in the MEK/ERK pathways was demonstrated in 22Rv1 cells by the silencing of c-Myc by means of short hairpin mRNA, which yielded effects resembling the targeting of MEK/ERK signaling. The clinically approved compound trametinib used in vitro yielded the same effects as U0126 on growth and C-Myc expression. Notably, U0126 and trametinib induced a drastic down-regulation of BMX, which is known to prevent apoptosis in cancer cells. The results of our study suggest that signal transduction-based therapy can, by disrupting the MEK/ERK/c-Myc axis, reduce human PCa radioresistance caused by increased c-Myc expression in vivo and in vitro and restores apoptosis signals.