N6-methyladenosine demethylase FTO enhances chemo-resistance in colorectal cancer through SIVA1-mediated apoptosis.

N6-methyladenosine (m6A) is the most pervasive RNA modification and is recognized as a novel epigenetic regulation in RNA metabolism. Although the m6A modification involves various physiological processes, its roles in drug resistance in colorectal cancer (CRC) still remain unknown. We analyzed the RNA expression profile of m6A/A (%) with MRM mass spectrometry in human 5-fluorouracil (5-FU)-resistant CRC tissues, and used the m6A RNA immunoprecipitation assay to validate the m6A-regulated target. Our results have shown that the m6A demethylase FTO was up-regulated in human primary and 5-FU-resistant CRC. Depletion of FTO decreased cell growth, colony formation and metastasis in 5-FU-resistant CRC cells in vitro and in vivo. Mechanistically, we identified SIVA1, a critical apoptotic gene, as a key downstream target of the FTO-mediated m6A demethylation. The m6A demethylation of SIVA1 at the CDS region induced its mRNA degradation via a YTHDF2-dependent mechanism. The SIVA1 levels were negatively correlated with the FTO levels in clinical CRC tissues. Notably, inhibition of FTO significantly reduced the tolerance of 5-FU in 5-FU-resistant CRC cells via the FTO-SIVA1 axis, whereas SIVA1-depletion could restore the m6A-dependent 5-FU sensitivity in CRC cells. In summary, our findings demonstrate a critical role of FTO as an m6A demethylase enhancing chemo-resistance in CRC cells, and suggest that FTO inhibition may restore the sensitivity of chemo-resistant CRC cells to 5-FU.

[SIVA1 Regulates the Stability of Single-Stranded DNA-Binding Protein 3 Isoforms].

The assembly of LIM-homeodomain (LIM-HD) transcriptional complex plays important roles in early neuronal development. The stability of LIM-HD is controlled by single-strand binding protein 3 (SSBP3) via a cascade mechanism protecting it from proteasomal degradation. The expression level of SSBP3 has to be precisely regulated. Although a decrease of SSBP3 level is associated with several diseases, the mechanism of SSBP3 downregulation and whether SSBP3 itself is subject to proteasomal degradation remain largely unknown. Two strongly conserved transcripts of the SSBP3 gene, SSBP3a and SSBP3c, were cloned from a human brain cDNA library. By RT-PCR, we show that Ssbp3c is continuously expressed in both embryonic and adult mouse brain, whereas Ssbp3a is restricted to embryonic brain tissue. By co-IP and GST pulldown assays, we identified SIVA1 as a novel SSBP3-binding factor. In a ubiquitination assay, we show that SIVA1 enhances the ubiquitination of SSBP3 and regulates its abundance. Our findings reveal the proteasomal degradation of SSBP3 for the first time and provide a rationale for an SIVAl-SSBP3-dependent mechanism for the disassembly of LIM-HD multiprotein complexes.

ANKHD1 silencing inhibits Stathmin 1 activity, cell proliferation and migration of leukemia cells.

ANKHD1 is highly expressed in human acute leukemia cells and potentially regulates multiple cellular functions through its ankyrin-repeat domains. In order to identify interaction partners of the ANKHD1 protein and its role in leukemia cells, we performed a yeast two-hybrid system screen and identified SIVA, a cellular protein known to be involved in proapoptotic signaling pathways. The interaction between ANKHD1 and SIVA was confirmed by co-imunoprecipitation assays. Using human leukemia cell models and lentivirus-mediated shRNA approaches, we showed that ANKHD1 and SIVA proteins have opposing effects. While it is known that SIVA silencing promotes Stathmin 1 activation, increased cell migration and xenograft tumor growth, we showed that ANKHD1 silencing leads to Stathmin 1 inactivation, reduced cell migration and xenograft tumor growth, likely through the inhibition of SIVA/Stathmin 1 association. In addition, we observed that ANKHD1 knockdown decreases cell proliferation, without modulating apoptosis of leukemia cells, while SIVA has a proapoptotic function in U937 cells, but does not modulate proliferation in vitro. Results indicate that ANKHD1 binds to SIVA and has an important role in inducing leukemia cell proliferation and migration via the Stathmin 1 pathway. ANKHD1 may be an oncogene and participate in the leukemia cell phenotype.

SIVA-1 interaction with PCBP1 serves as a predictive biomarker for cisplatin sensitivity in gastric cancer and its inhibitory effect on tumor growth in vivo.

Background: SIVA-1 has been reported to play a key role in cell apoptosis and gastric cancer (GC) chemoresistance in vitro. Nevertheless, the clinical significance of SIVA-1 in GC chemotherapy remains unclear. Methods and results: Immunohistochemistry and histoculture drug response assays were used to determine SIVA-1 expression and the inhibition rate (IR) of agents to GC and to further analyze the relationship between these two phenomena. Additionally, cisplatin (DDP)-resistant GC cells were used to elucidate the role and mechanism of SIVA-1 in vivo. The results demonstrated that SIVA-1 expression was positively correlated with the IR of DDP to GC but not with those of 5-fluorouracil (5-FU) or adriamycin (ADM). Furthermore, SIVA-1 overexpression with DDP treatment synergistically inhibited tumor growth in vivo by increasing PCBP1 and decreasing Bcl-2 and Bcl-xL expression. Conclusions: Our study demonstrated that SIVA-1 may serve as an indicator of the GC sensitivity to DDP, and the mechanism of SIVA-1 in GC resistance to DDP was preliminarily revealed.

SIVA-1 enhances acquired chemotherapeutic drug resistance of gastric cancer in vivo by regulating the ARF/MDM2/p53 pathway.

SIVA-1 has been shown to affect apoptotic processes in various different cell lines, and SIVA-1 significantly contributes to the decreased responsiveness of cancer cells to some chemotherapy agents. However, whether SIVA-1 has potential application in gastric cancer remains unknown. Therefore, the objective of this investigation was to clarify the distinct function of SIVA-1 in chemotherapeutic drug resistance within a living murine model with gastric malignancy, and initially elucidate the underlying mechanisms. In an established multidrug-resistant gastric cancer xenograft mouse model, lentivirus, named Lv-SIVA-1, was injected into xenograft tumors, and increased the mRNA and protein expression of endogenous SIVA-1 in tumors. Immunohistochemical assays of xenograft tumor showed that SIVA-1 was significantly upregulated, and the protein expression levels of SIVA-1 were highly increased, as detected by Western blotting. In addition, we detected the role of SIVA-1 in cell proliferation and cell apoptosis in gastric cancer cells by TUNEL and found that SIVA-1 decreased tumor cell apoptosis and promoted tumor growth in vivo. Using a TMT assay between tumor tissues of experimental and control groups, differentially expressed proteins were examined and three potential biomarkers of multidrug resistance (ARF, MDM2, and p53) were screened. We further investigated the molecular mechanism by which SIVA-1 played an efficient role against chemotherapies and found that overexpressed SIVA-1 leads to increased ARF and MDM2 expression and suppressed expression of p53 in tumor tissue. In conclusion, SIVA-1 plays a significant role in the multidrug resistance of gastric tumors. In addition, overexpressed SIVA-1 positively regulates cell proliferation, adjusts cycle progression, and reduces the response to drug treatment for gastric cancer in an ARF/MDM2/p53-dependent manner. This novel research provides a basis for chemical management of gastric cancer through regulation of SIVA-1 expression.

Inhibition of apoptosis-regulatory protein Siva-1 reverses multidrug resistance in gastric cancer by targeting PCBP1.

Introduction: Siva-1, as a pro-apoptotic protein, has been shown to induce extensive apoptosis in a number of different cell lines. In our previous study, we showed that overexpressed Siva-1 decreased the apoptosis of gastric cancer cells. So, we believe that it can also work as an anti-apoptotic protein. The present study aimed to determine the specific role of Siva-1 in anticancer drug resistance in gastric cancer in vivo and in vitro and preliminarily reveal the mechanism. Materials and Methods: A vincristine-resistant MKN-28/VCR gastric cancer cell line with stably downregulated Siva-1 was established. The effect of Siva-1 downregulation on chemotherapeutic drug resistance was assessed by measuring the IC50 and pump rate of doxorubicin. Proliferation, apoptosis of cells, and cell cycle were detected via colony formation assay and flow cytometry, respectively. Additionally, migration and invasion of cells was detected via wound healing and transwell assays. Moreover, we determined in vivo effects of LV-Siva-1-RNAi on tumor size, and apoptotic cells in tumor tissues were detected using TUNEL and hematoxylin and eosin staining. Results: Siva-1 downregulation reduced the pump rate of doxorubicin and enhanced the response to drug treatment. Siva-1 negatively regulated proliferation and promoted apoptosis of cells by potentiality G2-M phase arresting. Inhibition of Siva-1 expression in MKN-28/VCR cells significantly weakened wound healing ability and decreased invasion ability. Poly(C)-binding protein 1 (PCBP1) was identified as a Siva-1-interacting protein in yeast two-hybrid screening. Semiquantitative RT-PCR and western blotting revealed that Siva-1 downregulation could inhibit expression of PCBP1, Akt, and NF-κB and eventually decrease the expression of MDR1 and MRP1. Conclusion: he current study demonstrated that the downregulation of Siva-1, which functions as a regulator of MDR1 and MRP1 gene expression in gastric cancer cells by inhibiting PCBP1/Akt/NF-κB signaling pathway expression, enhanced the sensitivity of gastric cancer cells to certain chemotherapies.

Lentivirus-Mediated Overexpression of SIVA-1 Reverses Cisplatin Resistance in Gastric Cancer in vitro.

SIVA-1 plays a critical role in the induction of apoptosis in a number of different cell lines and participates in the mechanism of cisplatin (DDP)-mediated antitumor effects. However, the involvement of SIVA-1 in cisplatin resistance in gastric carcinoma has not been revealed. To explore the effect of SIVA-1 on DDP resistance, a recombinant pGV358-GFP-SIVA-1 lentiviral vector was constructed and transfected into human cisplatin-resistant MKN45/DDP gastric cancer cells. Subsequently, stable SIVA-1 overexpression was established in MKN45/DDP cells, which resulted in increased DDP sensitivity in MKN45/DDP cells in vitro. Flow cytometry demonstrated that SIVA-1 overexpression increased the percentage of apoptotic cells compared to that in the control. The colony formation assay clearly revealed that cell growth and proliferation were significantly suppressed following SIVA-1 overexpression. In addition, overexpression of SIVA-1 inhibited the migratory and invasive potential of MKN45/DDP cells in vitro. Western blot analysis indicated that SIVA-1 increased the expression levels of p53, p73, and p14ARF, whereas it reduced Bcl-2, MDM2, and Bcl-xL expression. In short, SIVA-1 upregulated the protein expression of p53, p73, and p14ARF and decreased that of Bcl-2, MDM2, and Bcl-xL in vitro and subsequently reversed cisplatin resistance in gastric cancer cells, suggesting that SIVA-1 serves as a valuable potential target for attenuating chemotherapy resistance.

Siva­1 regulates multidrug resistance of gastric cancer by targeting MDR1 and MRP1 via the NF­κB pathway.

Siva­1 is a well­known anti­apoptosis protein that serves a role in multiple types of cancer cells. However, whether Siva­1 affects multidrug resistance via the NF­κB pathway in gastric cancer is currently unknown. The present study aimed to determine the possible involvement of Siva­1 in gastric cancer anticancer drug resistance in vitro. A vincristine (VCR)­resistant KATO III/VCR gastric cancer cell line with stable Siva­1 overexpression was established. The protein expression levels of Siva­1, NF­κB, multidrug resistance 1 (MDR1) and multidrug resistance protein 1 (MRP1) were detected via western blotting. The effect of Siva­1 overexpression on anticancer drug resistance was assessed by measuring the 50% inhibitory concentration of KATO III/VCR cells to VCR, 5­fluorouracil and doxorubicin. The rate of doxorubicin efflux and apoptosis were detected by flow cytometry. Additionally, colony formation, wound healing and Transwell assays were used to detect the proliferation, migration and invasion of cells, respectively. The results of the current study revealed that the Siva­1­overexpressed KATO III/VCR gastric cancer cells exhibited a significantly decreased sensitivity to VCR, 5­fluorouracil and doxorubicin. The results of flow cytometry revealed that the percentage of apoptotic cells decreased following overexpression of Siva­1. The colony formation assay demonstrated that cell growth and proliferation were significantly promoted by Siva­1 overexpression. Additionally, Siva­1 overexpression increased the migration and invasion of KATO III/VCR cells in vitro. Western blot analysis determined that Siva­1 overexpression increased NF­κB, MDR1 and MRP1 levels. The current study demonstrated that overexpression of Siva­1, which functions as a regulator of MDR1 and MRP1 gene expression in gastric cancer cells via promotion of NF­κB expression, inhibited the sensitivity of gastric cancer cells to certain chemotherapies. These data provided novel insight into the molecular mechanisms of gastric cancer, and may be of significance for the clinical diagnosis and therapy of patients with gastric cancer.

Siva 1 Inhibits Cervical Cancer Progression and Its Clinical Prognosis Significance.

BACKGROUND: Cervical cancer is the second most common female malignancies. But the exact etiology of cervical cancer is still under investigation. Recent observations revealed that the loss expression of Siva 1 was related to several different types of tumors. It could play an indispensable role in both exogenous and endogenous apoptotic signaling pathways. Nevertheless, the relationship between Siva 1 expression and cervical cancer progression has not yet been fully clarified. This study aimed to explore the functional role of Siva1 in cervical cancer. MATERIALS AND METHODS: In this present experiment, expression of Siva 1 was detected in 87 cervical cancer, 34 CIN and 20 normal samples by immunohistochemistry. The correlation of Siva 1 expression and overall survival times (OS) was analyzed by Kaplan-Meier analysis. We up-regulated the expression of Siva 1 by plasmid pCMV3-Siva 1 in C33A cells. CCK8, flow cytometry, wound-healing, and transwell assays were performed to examine the influences of Siva 1 expression on cell proliferation, apoptosis, migration and invasion. RESULTS: The expression of Siva 1 was decreased in cervical cancer tissues compared with CIN and normal tissues. In addition, the Siva 1 immunoreactivity was significantly associated with tumor differentiation. Patients with Siva 1 negative staining exhibited a significantly decreased overall survival. Then, we established stable Siva 1 ectopic expression cells, and we found that elevated expression of Siva 1 promoted apoptosis, inhibited proliferation, and suppressed migration and invasion of cervical cancer cells. CONCLUSION: The present study revealed a crucial role of Siva 1 in tumor progression and it may be a valuable prognostic indicator of cervical cancer.

Siva-1 emerges as a tissue-specific oncogene beyond its classic role of a proapoptotic gene.

Siva-1 is a typical apoptotic protein commonly activated by the p53 tumor suppressor protein and should therefore participate in a barrier against the development of cancer. It has proapoptotic activities in various cell systems. Recent findings suggest that Siva-1 possesses several other apoptosis-independent functions and interacts with many other proteins not directly involved in apoptosis. It harbors the ARF E3 ubiquitin protein ligase activity, a property that is clearly prooncogenic and leads to p53 degradation through the upregulation of the Hdm2 protein level. Surprisingly, recent evidence shows that Siva-1 absence prevents the development of non-small cell lung carcinomas in a mouse model and reveals the oncogenic roles in the same types of human cells, indicating its unique function as an oncogene in the cell context-dependent manner. Herein, we review reported activities of Siva-1 in various experimental settings and comment on its ambiguous function in tumor biology.

Siva 1 inhibits proliferation, migration and invasion by phosphorylating Stathmin in ovarian cancer cells.

Ovarian cancer is one of the most common types of gynecologic malignant tumor, with high incidence and high mortality rates. It is difficult to diagnose ovarian cancer early due to the complex structure and function of the ovaries. Siva 1 is a well-known pro-apoptosis protein that functions in multiple types of cancer cells: There are several studies demonstrating that Siva 1 arrests apoptosis and facilitates cancer development in osteosarcoma and non-small cell lung cancer. Whether Siva 1 functions in ovarian cancer remains unknown. In the present study, it was established that Siva 1 was stably overexpressed in ovarian cancer cell lines, and demonstrated that the overexpression of Siva 1 inhibited proliferation, promoted apoptosis and suppressed migration and invasion by facilitating phosphorylation of Stathmin and polymerization of α-tubulin in ovarian cancer cells. These data provide specific novel insights into the molecular mechanism of ovarian cancer, and may be of significance for the clinical diagnosis and therapy of ovarian cancer.

Anticancer activity of recombinant Siva1 protein in human nasopharyngeal carcinoma cell line CNE-2.

AIMS: To clone and express Siva1 protein, and to investigate the role of Siva1 protein in proliferation, apoptosis, invasion, and migration of human nasopharyngeal carcinoma cell line CNE-2 in vitro and in vivo. METHODS: The PCR fragment of Siva1 from human nasopharyngeal carcinoma cell line CNE-2 were double digested with BamHI and SalI and then induced into the pQE30 vector double digested by the same enzymes. The pQE30 vector harboring Siva1 was introduced into M15 competent cells and then induced by isopropyl ß -D-1-thiogalactopyranoside (IPTG). The Siva1 fusion protein was identified by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then separated and purified by Ni-affinity chromatography. Subsequently, the effects of recombinant Siva1 protein on proliferation, apoptosis, invasion and migration were assayed in vitro and in vivo. RESULTS: The transformed cells expressed Siva1 fusion protein with a molecular weight of approximately 12 kDa. Cell counting kit-8 (CCK-8) assay showed that the Siva1 protein significantly inhibited the proliferation of the CNE-2 cells at a concentration of 10 µ mol/L. In addition, compared to the control, the Siva1 protein promoted the apoptosis of the cancer cells. And, the Siva1 protein greatly suppressed the invasion and migration of the cancer cells. In vivo, the Siva1 protein significantly inhibited the tumor growth of the tumor-bearing mice. Further, the Siva1 treatment markedly upregulated Bax, caspase-3, and downregulated Bcl-2 protein levels in the transplanted tumor tissue. CONCLUSIONS: The Siva1 protein has a significant anticancer activity on human nasopharyngeal carcinoma cell line CNE-2 including inhibiting proliferation, invasion, migration and promoting apoptosis of the cancer cells.

SIVA, a target of p53, is downregulated in myelodysplastic syndromes

Background: SIVA is a transcriptional target of p53 that plays a potential role in the development and progression of cancer. In this study, we analyzed SIVA1 and SIVA2 expression, and its association with clinical features and TP53 and MDM2 expression in bone marrow cells from healthy donors and myelodysplastic syndrome (MDS) patients. Methods: Fifty-five untreated patients with MDS and 22 healthy donors were included. Gene expression was evaluated by quantitative PCR. For statistical analysis, Mann­Whitney test, Spearman correlation analysis and Log-rank (Mantel-Cox) were used, as appropriate. A p value <0.05 was considered statistically significant. Results: SIVA1 and SIVA2 transcripts were significantly decreased in bone marrow samples from MDS patients compared to healthy donors, and positively correlated with MDM2 and TP53 expression in MDS patients (all p < 0.05). MDM2 expression was also downregulated in bone marrow samples from MDS patients compared to healthy donors (p < 0.05). However, SIVA1, SIVA2, MDM2 and TP53 expressions did not impact on MDS outcomes. Conclusions: SIVA1 and SIVA2 transcripts are downregulated in bone marrow samples from MDS patients (AU)

The N-terminal 33 amino acid domain of Siva-1 is sufficient for nuclear localization

Siva-1 induces apoptosis in multiple pathological processes and plays an important role in the suppression of tumor metastasis, protein degradation, and other functions. Although many studies have demonstrated that Siva-1 functions in the cytoplasm, a few have found that Siva-1 can relocate to the nucleus. In this study, we found that the first 33 amino acid residues of Siva-1 are required for its nuclear localization. Further study demonstrated that the green fluorescent protein can be imported into the nucleus after fusion with these 33 amino acid residues. Other Siva-1 regions and domains showed less effect on Siva-1 nuclear localization. By site-mutagenesis of all of these 33 amino acid residues, we found that mutants of the first 1-18 amino acids affected Siva-1 nuclear compartmentalization but could not complete this localization independently. In summary, we demonstrated that the N-terminal 33 amino acid residues were sufficient for Siva-1 nuclear localization, but the mechanism of this translocation needs additional investigation.

Two-way Regulation of Apoptosis-inducing Factor SIVA-1 in the Proliferation and Differentiation of Osteoblasts under Fluid Shear Stress / 中国微创外科杂志

Objective To investigate the regulatory effect of fluid shear stress(FSS) on the proliferation and differentiation of osteoblasts,as well as the expression of apoptosis-inducing factor,SIVA-1.Methods The third-passage osteoblasts were divided into five experiment groups and one control group.In the experiment groups,1.2 Pa FSS were given to the osteoblasts for 0.25,0.5,1,2,and 4 hours respectively,while the control group received no FSS.Afterwards,the cells were harvested to measure MTT value and ALP activity;mRNA level of SIVA-1 were determined by RT-PCR.Results MTT revealed that the cells proliferation markedly increased in the 0.25 h and 0.5 h experiment groups with advanced cell growth curve;whereas significantly inhibited in 1,2,and 4 h groups.The FSS also increased the ALP activity at 0.25 and 0.5 hour,especially in the 0.5 h group(2.4320?0.205 S unit/100ml,158% of the control;P