SiRNF8 Delivered by DNA Framework Nucleic Acid Effectively Sensitizes Chemotherapy in Colon Cancer.

Background: The evident side effects and decreased drug sensitivity significantly restrict the use of chemotherapy. However, nanoparticles based on biomaterials are anticipated to address this challenge. Methods: Through bioinformatics analysis and colon cancer samples, we initially investigated the expression level of RNF8 in colon cancer. Next, we constructed nanocarrier for delivering siRNF8 based on DNA tetrahedron (si-Tet), and Doxorubicin (DOX) was further intercalated into the DNA structure (si-DOX-Tet) for combination therapy. Further, the effects and mechanism of RNF8 inhibition on the sensitivity of colon cancer cells to DOX chemotherapy have also been studied. Results: RNF8 expression was increased in colon cancer. Agarose gel electrophoresis, transmission electron microscopy, and size distribution and potential analysis confirmed the successful preparation of the two nanoparticles, with particle sizes of 10.29 and 37.29 nm, respectively. Fluorescence imaging reveals that the carriers can be internalized into colon cancer cells and escape from lysosomes after 12 hours of treatment, effectively delivering siRNF8 and DOX. Importantly, Western blot analysis verified treatment with 50nM si-Tet silenced RNF8 expression by approximately 50% in colon cancer cells, and combined treatment significantly inhibited cell proliferation. Furthermore, the CCK-8 assay demonstrated that si-Tet treatment enhanced the sensitivity of colon cancer cells to the three chemotherapeutic drugs. Significant more DNA damage was detected after treatment with both si-Tet or si-DOX-Tet. Further flow cytometry analysis revealed that si-DOX-Tet treatment led to significantly more apoptosis, approximately 1.6-fold higher than treatment with DOX alone. Mechanistically, inhibiting RNF8 led to decreased ABCG2 expression and DOX efflux, but increased DNA damage, thereby enhancing the chemotherapeutic effect of DOX. Conclusion: We have successfully constructed si-DOX-Tet. By inhibiting the expression of RNF8, it enhances the chemotherapy sensitivity of DOX. Therefore, this tetrahedral FNA nanocarrier offers a new approach for the combined treatment of colon cancer.

Carbon ion irradiation induces DNA damage in melanoma and optimizes the tumor microenvironment based on the cGAS-STING pathway.

PURPOSES: Increased number of studies reveal the crucial role of the Cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway in anti-tumor immunity. In this study, we aim to explore the effect of cGAS/STING on tumor immune microenvironment of melanoma after carbon ion radiotherapy (CIRT) and the underlying mechanism. METHODS: C57BL/6 mouse tumor models were used to evaluate the efficacy of different treatments (X-ray, carbon ion, PD-L1 inhibitor and combination therapies) on tumor growth and process. Mass cytometry was performed to assess tumor-infiltrating lymphocytes (TILs). DNA damage response (DDR) and cGAS/STING pathway were investigated by immunofluorescence-co-localization assays, γ-H2AX, P53-binding protein 1 (53BP1), Breast Cancer 1 (BRCA1), and cGAS measurements. RESULTS: Carbon ion irradiation caused more DNA damages and cGAS-STING pathway activation compared with X-ray irradiation, and the former slowed the melanoma growth in syngeneic model. Although X-ray irradiation is not sensitive for melanoma treatment, carbon ion irradiation showed a significant anti-tumor effect for melanoma treatment. TILs analysis revealed that CIRT boosted the infiltration of natural killer (NK), CD4+, and CD8+ T cells, meanwhile increased the number of immune checkpoint (programmed death-1, PD-1, lymphocyte activation gene 3, LAG-3 and T-cell immunoglobulin and mucin domain-containing protein 3, TIM-3). Moreover, CIRT increased PD-L1 exposure on cell surface compared with X-ray group. Furthermore, CIRT combined with PD-L1 inhibitor therapy increased the number of T cells and NK cells in melanoma, and slowed the growth of melanoma compared with other therapies. CONCLUSIONS: Our findings showed that CIRT displayed biological effects by increasing DNA damages of tumor cells and improving immunity in melanoma, which indicated that CIRT might be a potential synergetic treatment for radiotherapy and radioimmunotherapy in melanoma patients. Our works put forward a new insight to provide an effective strategy for melanoma therapy. These findings may help in the design of strategies on melanoma in clinical studies.

Downregulation of SOX9 suppresses breast cancer cell proliferation and migration by regulating apoptosis and cell cycle arrest.

SRY-related high-mobility group box 9 (SOX9) is an important transcriptional factor that regulates diverse genes involved in development and stemness. Dysregulation of SOX9 encourages carcinogenesis in various types of cancer, including breast cancer. The present study aimed to explore the role of SOX9 in triple-negative breast cancer (TNBC). SOX9 expression was significantly upregulated in the TNBC MDA-MB-231, MDA-MB-436 and MDA-MB-468 cell lines compared with that in BT-549 cells. Based on a lentivirus assay, SOX9 inhibition in MDA-MB-231 and MDA-MB-436 cells suppressed cell proliferation and colony formation. Apoptosis was increased and the cell cycle was arrested at the G0/G1 phase in SOX9-knockdown cells. Transwell and wound-healing assays demonstrated that SOX9 inhibition decreased the migration and invasion of MDA-MB-231 and MDA-MB-436 cells. RNA sequencing identified that numerous genes were regulated by SOX9, including nucleophosmin, thioredoxin reductase 1, succinate dehydrogenase complex subunit D, nuclear receptor binding SET domain protein 2, eukaryotic translation initiation factor 4γ1 and glycogen phosphorylase L. Overall, the current study suggested that SOX9 acted as an oncogene in TNBC.

RAD6B Plays a Critical Role in Neuronal DNA Damage Response to Resist Neurodegeneration.

RAD6 participates in DNA double-strand breaks (DSBs) repair by ubiquitinating histone H2B in mitotic cells. In terminally differentiated cells, however, the mechanisms of DNA damage repair are less well known. In this study, we investigate whether RAD6B is involved in DSBs repair in neurons and effects of RAD6B deficiency on neuronal survival. We compared neurons of RAD6B-deficient mice with those of littermate wild type (WT) mice and induced DNA damage by X-ray irradiation. We provide evidence that RAD6B is essential for neural DDR and RAD6B deficiency results in increased genomic instability and neurodegeneration. Moreover, higher levels of p53 and p21 are present in the brains of RAD6B-deficient mice, which may be responsible for neuronal senescence, and degeneration. In addition, behavioral experiments show that RAD6B-deficient mice exhibit marked learning and memory deficits. In conclusion, these findings suggest that RAD6B is critical for neural integrity and that the absence of RAD6B accelerates neurodegeneration in mice.

DNA damage preceding dopamine neuron degeneration in A53T human α-synuclein transgenic mice.

Defective DNA repair has been linked with age-associated neurodegenerative disorders. Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by genetic and environmental factors. Whether damages to nuclear DNA contribute to neurodegeneration of PD still remain obscure. in this study we aim to explore whether nuclear DNA damage induce dopamine neuron degeneration in A53T human α-Synuclein over expressed mouse model. We investigated the effects of X-ray irradiation on A53T-α-Syn MEFs and A53T-α-Syn transgene mice. Our results indicate that A53T-α-Syn MEFs show a prolonged DNA damage repair process and senescense phenotype. DNA damage preceded onset of motor phenotype in A53T-α-Syn transgenic mice and decrease the number of nigrostriatal dopaminergic neurons. Neurons of A53T-α-Syn transgenic mice are more fragile to DNA damages.

Fiber-modified adenovirus-mediated suicide gene therapy can efficiently eliminate bladder cancer cells in vitro and in vivo.

Adenovirus-mediated gene therapy is a promising strategy for bladder cancer treatment. However, the loss of the coxsackie and adenovirus receptor (CAR) in bladder cancer cells decreases the infection efficiency of the therapeutic adenovirus. In this study, we constructed an Arg-Gly-Asp (RGD)-modified adenovirus, RGDAd-UPII-TK, that carries a suicide gene called HSV-TK that is driven by a human UPII promoter. Then, we tested the bladder cancer specificity of the UPII promotor and the expression of the HSV-TK protein. Additionally, we observed a potent cytotoxic effects of RGDAd-UPII-TK and ganciclovir (GCV) on bladder cancer as demonstrated by reduced cell survival and morphology changes in vitro. Furthermore, we confirmed that RGDAd-UPII-TK in combination with a GCV injection could significantly reduce the established T24 tumor growth and increase apoptosis in vivo. Altogether, our results indicated that the recombinant adenovirus RGDAd-UPII-TK could target bladder cancer through valid gene therapy.

DNA Damage-Induced Foci of E2 Ubiquitin-Conjugating Enzyme are Detectable upon Co-transfection with an Interacting E3 Ubiquitin Ligase.

DNA damage repair elements accumulate at DNA damage sites to form ionizing radiation-induced foci (IRIF) for damage repair. IRIF, which represent direct evidence of DNA damage response activity, which are conveniently to be observed via immunofluorescence staining. Protein ubiquitination plays an important role in initiating the DNA damage response. Following DNA damage, the substrate binding protein E3 ubiquitin-ligases enzymes are recruited to DNA damage sites, then the E2 ubiquitin-conjugating enzymes are recruited to these sites by the E3 where they catalyze protein ubiquitination. However, IRIF of E2 enzymes are relatively transient and unstable in vivo and difficult to detect. Here, we present a new method for the observation of E2 IRIF. This method is based on the co-transfection of interacting E2 and E3 enzymes into cells and identifies IRIF via immunofluorescence following DNA damage.

Adenovirus-mediated downregulation of the ubiquitin ligase RNF8 sensitizes bladder cancer to radiotherapy.

The ubiquitin ligase RNF8 promotes the DNA damage response (DDR). We observed that the expression of RNF8 was increased in bladder cancer cells and that this change in RNF8 expression could be reversed by adenovirus-mediated shRNA treatment. Moreover, we found that RNF8 knockdown sensitized bladder cancer cells to radiotherapy, as demonstrated by reduced cell survival. Additionally, the absence of RNF8 induced a high rate of apoptosis and impaired double-strand break repair signaling after radiotherapy. Furthermore, experiments on nude mice showed that combining shRNF8 treatment with radiotherapy suppressed implanted bladder tumor growth and enhanced apoptotic cell death in vivo. Altogether, our results indicated that RNF8 might be a novel target for bladder cancer treatment.

RNF8 deficiency results in neurodegeneration in mice.

The progressive loss of neurons causes neurodegenerative diseases. Because the accumulation of DNA breaks results in neuronal apoptosis, the lack of a variety of DNA damage repair-related proteins contributes to neurodegeneration. The ubiquitin ligase RNF8 plays an important role in DNA double-strand break repair via histone ubiquitination. However, the function of RNF8 in terminally differentiated neurons remains unknown. This study aimed to determine whether RNF8 is involved in the DNA damage response in neurons and contributes to neurodegeneration. Here, we present evidence suggesting that RNF8 deficiency results in DNA damage accumulation and neuronal apoptosis. RNF8(-/-) mice exhibit neuronal degeneration and reactive astrocytosis. Neurons from RNF8(-/-) mice appear to be more susceptible to X-ray-induced DNA damage. These changes were consistent with the behavioral performances of the RNF8-deficient mice, which included impaired performances in the open-field test and step-down avoidance task. Overall, these findings show that RNF8 is required for DNA damage repair in neurons. RNF8 deficiency is sufficient to cause neuronal pathology and cognitive decline, and the loss of RNF8 results in neuron degeneration.

Inhibitory effect of valproic acid on bladder cancer in combination with chemotherapeutic agents in vitro and in vivo.

Histone deacetylase inhibitors (HDACIs) are a promising class of drugs that act as antiproliferative agents by promoting differentiation and inducing apoptosis. Valproic acid (VPA) is an HDACI that has been widely used as an anti-convulsant and shows promise as a chemotherapeutic drug for a number of tumor cells. The present study aimed to investigate the inhibitory effect of VPA on the viability of bladder cancer cells and its synergistic effect with chemotherapeutic agents in vitro and in vivo. The cell viability of human bladder cancer cell lines following treatment with VPA and/or VPA in combination with mitomycin C, cisplatin (DDP) and adriamycin were determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Hoechst staining was used to observe the morphology of the apoptotic cells. Survivin protein and acetylated histone H3 levels were quantified using western blot analysis. The in vivo tumor growth inhibition of VPA was determined in rats with N-methyl-N-nitrosourea-induced bladder cancer. VPA significantly inhibited the growth of the bladder cancer cells in a concentration- and time-dependent manner. Furthermore, improved results were achieved for tumor inhibition when VPA was combined with chemotherapeutic agents in vitro and in vivo. Survivin expression decreased and acetylated histone H3 expression increased in the bladder cancer cells following the treatment with VPA. Intravesical injections of VPA were able to inhibit tumor progression when combined with DDP. In conclusion, VPA acts as an HDACI that has a direct anticancer effect and markedly enhances the action of several chemotherapy agents. VPA may sensitize bladder cancer to anticancer drugs by downregulating survivin expression.

Two hour exposure to sodium butyrate sensitizes bladder cancer to anticancer drugs.

OBJECTIVES: To investigate the inhibitory effect of sodium butyrate (NaB) on the proliferation of human bladder cancer cell lines and its synergetic effect with anticancer drugs in treating bladder cancer in vitro and in vivo. METHODS: The inhibitory effects of NaB on human bladder cancer cell lines in vitro and the synergetic effect of NaB with mitomycin c, cisplatin (CDDP) and adriamycin were detected by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. Hoechst staining and electron microscopy were used to observe morphology for apoptotic cells after NaB treatment. Fas, bcl-2 and caspase-3 were determined with flow cytometry. In vivo synergetic effects were detected in N-methyl-N-nitrosourea induced bladder cancer model rats. RESULTS: NaB significantly inhibited the growth of bladder cancer cell lines in a concentration and time dependent manner. Better results of tumor inhibition have been achieved when NaB was combined with CDDP, mitomycin c and adriamycin, rather than used alone. Furthermore, 2 h exposure to NaB can sensitize bladder cancer to chemotherapy agents. The Bcl-2 expression in bladder cancer cells is decreased and caspase-3 expression increased after NaB treatment. Intravesical application of NaB combined with CDDP can significantly inhibit tumor growth and progression. CONCLUSIONS: NaB has a direct anticancer effect and can markedly enhance the action of several chemotherapy agents. 2 h expose to NaB can also sensitize bladder cancer to anticancer drugs. NaB may be an excellent candidate agent for intravesical application in treating bladder cancer.