Anti-cancer activity of secreted aspartyl proteinase protein from Candida tropicalis on human cervical cancer HeLa cells.

Cervical cancer is the fourth leading cause of cancer-related death in women worldwide. Microbial products are valuable sources of anti-cancer drugs. The aim of this study was to isolate secreted aspartyl proteinase protein from Candida tropicalis, investigate its inhibitory effect on human cervical cancer HeLa cells, and analyze the expression profiling of selected nuclear stem cell-associated transcription factors. The presence of secreted aspartyl proteinase protein was confirmed by the expression of SAP2 and SAP4 genes in C. tropicalis during the yeast-hyphae transition phase. The enzyme was purified and characterized using the aqueous two-phase system purification method, as well as proteolytic activity and the Bradford and micro-Kjeldahl methods, respectively. The in vitro anti-cancer properties of secreted aspartyl proteinase protein were evaluated by MTT assay, microscopic image analysis, nitric oxide (NO) scavenging activity assay, intracellular reactive oxygen species (ROS) production assay, and RT-qPCR. The isolated C. tropicalis secreted aspartyl proteinase protein exhibited proteinase activity with values ranging from 93.72 to 130.70 µg/mL and 89.88 to 127.72 µg/mL according to the Bradford and micro-Kjeldahl methods, respectively. Secreted aspartyl proteinase showed effective cytotoxicity in HeLa cell line leading to significant morphological changes. Additionally, it exhibited increased free radical scavenging activity compared to the untreated control group, as evidenced by nitrite inhibition. ROS production increased in HeLa cells exposed to secreted aspartyl proteinase. The expression levels of the nuclear stem cell-associated transcription factors octamer-binding transcription factor 4 (OCT4), sex determining region Y-box 2 (SOX2), and Nanog homeobox (NANOG) were significantly downregulated in the HeLa cells treated with secreted aspartyl proteinase. Secreted aspartyl proteinase protein may be a promising anti-cancer agent, as it effectively affects gene expression and may ultimately reduce the development and progression of cervical cancer. Targeting the genes related to nuclear stem cell-associated transcription factors may provide a novel amenable to cancer treatment.

Pseudogene OCT4-pg5 upregulates OCT4B expression to promote bladder cancer progression by competing with miR-145-5p.

Bladder cancer (BC) is one of the most common malignant neoplasms worldwide. Competing endogenous RNA (ceRNA) networks may identify potential biomarkers associated with the progression and prognosis of BC. The OCT4-pg5/miR-145-5p/OCT4B ceRNA network was found to be related to the progression and prognosis of BC. OCT4-pg5 expression was significantly higher in BC cell lines than in normal bladder cells, with OCT4-pg5 expression correlating with OCT4B expression and advanced tumor grade. Overexpression of OCT4-pg5 and OCT4B promoted the proliferation and invasion of BC cells, whereas miR-145-5p suppressed these activities. The 3' untranslated region (3'UTR) of OCT4-pg5 competed for miR-145-5p, thereby increasing OCT4B expression. In addition, OCT4-pg5 promoted epithelial-mesenchymal transition (EMT) by activating the Wnt/ß-catenin pathway and upregulating the expression of matrix metalloproteinases (MMPs) 2 and 9 as well as the transcription factors zinc finger E-box binding homeobox (ZEB) 1 and 2. Elevated expression of OCT4-pg5 and OCT4B reduced the sensitivity of BC cells to cisplatin by reducing apoptosis and increasing the proportion of cells in G1. The OCT4-pg5/miR-145-5p/OCT4B axis promotes the progression of BC by inducing EMT via the Wnt/ß-catenin pathway and enhances cisplatin resistance. This axis may represent a therapeutic target in patients with BC.

Ectopic expression of OCT4B1 Decreases Fertility Rate and Changes Sperm Parameters in Transgenic Mice.

Background: The octamer-binding transcription factor-4 (OCT4) is known as an established important regulator of pluripotency, as well as a genetic "master switch" in the self-renewal of embryonic stem and germ cells. OCT4B1, one of the three spliced variants of human OCT4, plays crucial roles in the regulation of pluripotency and stemness. Objectives: The present study developed a transgenic mouse model containing an OCT4B1-expressing construct under the transcriptional direction of mouse mammary tumor virus promoter (pMMTV) to evaluate the role of OCT4B1 in the function of male germ cells in terms of fertility potential. Additionally, the effect of ectopic OCT4B1 overexpression on endogenous OCT4 expression was examined in mouse embryonic stem cells (mESCs). Material and Methods: The pMMTV-OCT4B1cDNA construct was injected into the pronuclei of 0.5-day NMRI embryos. Transgenic mice were identified based on the PCR analysis of tail DNA. Further, Diff-Quik staining was applied to assess sperm morphology, while the other sperm parameters were analyzed through a conventional light microscopic evaluation according to World Health Organization (WHO) criteria. The fertility rate was scored by using in vitro frtilization (IVF) method. Furthermore, mESCs was electroporated with the OCT4B1cDNA-containing constructs, followed by analyzing through employing semi-quantitative RT-PCR and western blotting. Results: The results demonstrated the changes in sperm morphology, as well as a statistically significant decrease in the other sperm parameters (count, viability, and motility) and fertility rate (p<0.05) in the transgenic mice compared with the control group. The assessment of the cause of the embryonic stem cell (ESC) death following transfection revealed a significant reduction in the endogenous OCT4 expression at both mRNA and protein levels in the transfected mESCs compared to the control ones. Conclusion: In general, the in vivo results suggested a potential role of OCT4B1 in the spermatogenesis process. These results represented that the overexpression of OCT4B1 may induce its role in spermatogenesis and fertility rate by interfering endogenous OCT4 expression. However, further studies are required to clarify the mechanisms underlying OCT4B1 function.

Novel Variant of OCT4, Named OCT4B5, is Highly Expressed in Human Pluripotent Cells.

Alternative promoter and alternative splicing are two important mechanisms of gene regulation and protein diversity in different physiological contexts of eukaryotes, especially in stem cells and developmental stages. Pou5f1 gene which codes the stemness marker OCT4, utilizes alternative splicing and promoter mechanisms, which result in generation of multiple spliced variants and subsequently multiple protein isoforms. By far, nine variants of OCT4 (OCT4A, OCT4B, OCT4B1, OCT4B2, OCT4B3, OCT4B4, OCT4C, OCT4C1, and OCT4D) have been introduced. It has been well established that OCT4A plays essential roles in early developmental stages as well as maintenance of stemness in embryonic stem cells (ESCs). However, the roles and functions of other variants and isoforms of OCT4 in biological systems are less appreciated. In this study, we report a new OCT4 variant, designated as OCT4B5. RT-PCR assay on different human cell lines including pluripotent, normal and cancer cells showed that OCT4B5 is expressed at variable level in different cell lines. By semi-quantifying of OCT4B5 expression in pluripotent and differentiated states of NT2 cell lines, we reveal that this variant of OCT4 is highly expressed in undifferentiated state and its expression is down-regulated upon differentiation. Compared to OCT4A which is sharply down-regulated in retinoic acid induced differentiation of NT2 cell line, the expression of OCT4B5 remains at low level in differentiated state. Overall, this study emphasizes the complexity of OCT4 gene expression and regulation in different states of stem cells and physiological contexts. Graphical Abstract.

OCT4B1 Promoted EMT and Regulated the Self-Renewal of CSCs in CRC: Effects Associated with the Balance of miR-8064/PLK1.

Cancer stem cells (CSCs) are the main cause of tumor generation, recurrence, metastasis, and therapy failure in various malignancies including colorectal cancer (CRC). Accumulating evidence suggests that tumor cells can acquire CSC characteristics through the epithelial-mesenchymal transition (EMT) process. However, the molecular mechanism of CSCs remains unclear. OCT4B1 is a transcript of OCT4, which is initially expressed in embryonic stem and carcinoma cells, and is involved in the regulation and maintenance of an undifferentiated state of stem cells. In this study, three-dimensional (3D) microspheres were confirmed as CRC stem cells. Compared with that of parental cells, their self-renewal ability was significantly increased, and OCT4B1 expression was increased and promoted the EMT process. The knockdown of OCT4B1 decreased the self-renewal of CSCs and reversed EMT. Moreover, OCT4B1 induced the expression of Polo-like kinase 1 (PLK1), which is a key regulator of EMT in tumor cells. Further examination showed that OCT4B1 regulated the miR-8064/PLK1 balance to exert its function. Taken together, our data suggest that OCT4B1 may be involved in regulating the self-renewal of colorectal CSCs through EMT, which is at least partially due to the miR-8064/PLK1 balance. This study indicates that OCT4B1 is a potential therapeutic target for CRC by targeting CSCs.

OCT4B-190 protects against ischemic stroke by modulating GSK-3ß/HDAC6.

OCT4 is a key regulator in maintaining the pluripotency and self-renewal of embryonic stem cells (ESCs). Human OCT4 gene has three mRNA isoforms, termed OCT4A, OCT4B and OCT4B1. The 190-amino-acid protein isoform (OCT4B-190) is one of the major products of OCT4B mRNA, the biological function of which is still not well defined. Recent evidence suggests that OCT4B-190 may function in the cellular stress response. The glycogen synthase kinase-3ß (GSK-3ß) and histone deacetylase 6 (HDAC6) are also key stress modulators that play critical roles in the ischemic cascades of stroke. Hence, we here further investigated the effects of OCT4B-190 in the experimental stroke, and explored the underlying roles of GSK-3ß and HDAC6. We found that OCT4B-190 overexpression enhanced neuronal viability at 24 h after oxygen-glucose deprivation (OGD) treatment. Moreover, in male C57BL/6 mice subjected to transit middle cerebral artery occlusion (MCAO), OCT4B-190 overexpression reduced infarct volume and improved neurological function after stroke. Notably, we found spatio-temporal alterations of GSK-3ß and HDAC6 in the ischemic cortex and striatum, which were affected by adenovirus-mediated OCT4B-190 overexpression. OCT4B-190 demonstrated similar impacts on neuronal cultures in vitro, downregulating OGD-induced GSK-3ß activity and HDAC6 expression. In addition, we found that GSK-3ß and HDAC6 were co-expressed in the cytoplasm of neurons, and OCT4B-190 had an effect on interactions between GSK-3ß and HDAC6 in neuronal cultures subjected to OGD treatment. These findings suggest that OCT4B-190 exerts neuroprotection in the experimental stroke potentially by regulating actions of GSK-3ß and HDAC6 simultaneously, which may be an attractive therapeutic strategy for ischemic stroke.

OCT4B Isoform Promotes Anchorage-Independent Growth of Glioblastoma Cells.

OCT4, also known as POU5F1 (POU domain class 5 transcription factor 1), is a transcription factor that acts as a master regulator of pluripotency in embryonic stem cells and is one of the reprogramming factors required for generating induced pluripotent stem cells. The human OCT4 encodes three isoforms, OCT4A, OCT4B, and OCT4B1, which are generated by alternative splicing. Currently, the functions and expression patterns of OCT4B remain largely unknown in malignancies, especially in human glioblastomas. Here, we demonstrated the function of OCT4B in human glioblastomas. Among the isoform of OCT4B, OCT4B-190 (OCT4B19kDa) was highly expressed in human glioblastoma stem cells and glioblastoma cells and was mainly detected in the cytoplasm rather than the nucleus. Overexpression of OCT4B19kDa promoted colony formation of glioblastoma cells when grown in soft agar culture conditions. Clinical data analysis revealed that patients with gliomas that expressed OCT4B at high levels had a poorer prognosis than patients with gliomas that expressed OCT4B at low levels. Thus, OCT4B19kDa may play a crucial role in regulating cancer cell survival and adaption in a rigid environment.

OCT4B2, a novel alternative spliced variant of OCT4, is significantly upregulated under heat-stress condition and downregulated in differentiated cells.

OCT4 is a crucial transcription factor that maintains self-renewal and pluripotency of embryonic stem and embryonic carcinoma cells. The human OCT4 gene can generate at least three variants (OCT4A, OCT4B, and OCTB1) via alternative splicing and alternative promoters. It has been previously reported that OCT4A is the main isoform, retaining stemness state in embryonic stem and embryonic carcinoma cells. There are several reports on the expression of OCT4A, OCT4B, and OCT4B1 in some cancers and tumor cells. The expression of OCT4 in cancer tissues and cell lines appeared to be highly controversial since it was believed that OCT4 is exclusively expressed in embryonic stem/embryonic carcinoma cells. Here, we are reporting the detection of a novel alternatively spliced variant of OCT4, OCT4B2, in several pluripotent and tumor cell lines. Moreover, the expression pattern of OCT4B2 in the course of neural differentiation of NT2 and NCCIT, embryonic carcinoma cells, was similar to that of OCT4A. OCT4B2 was highly expressed in undifferentiated cells; however, its expression was sharply downregulated upon induction of differentiation. Overexpression of OCT4B2 did not affect the distribution of cells in different cell-cycle phases of transfected cells, compared to the mock transfected cells. Interestingly, the expression of OCT4B2 transcript was elevated under the heat-shock induction. In conclusion, we are reporting a new variant of OCT4, which is expressed under different physiological conditions. The finding shed more light on complexity of OCT4 expression and functions.

Novel variant of OCT4B4 is differentially expressed in human embryonic stem and embryonic carcinoma cells.

POU domain proteins are an important family of transcription factors that regulates cell type-specific gene expression. One of the most crucial members of this family that maintains pluripotency and self-renewal of embryonic stem cells is POU5F1/OCT4. The OCT4 gene can generate several variants under different situations/cell types includes OCT4A that is the major factor sustains pluripotency in embryonic stem and embryonic carcinoma cells, and also OCT4B and OCT4B1, which are transcribed from a different potential promoter located in intron1 and are expressed in various tissues and cell types. In present study, during expression check of OCT4B1 in embryonic carcinoma cells (NT2), we discovered a novel OCT4 transcript for the first time and designated it as OCT4B4. This variant is expressed in various human pluripotent cells and its expression is down-regulated upon induction of differentiation. Moreover, knocking down of OCT4B4 by shRNA resulted in increased accumulation of transfected cells in G0/G1 phase compared to the mock-transfected control cells.

Down-regulation of anti-apoptotic genes in tumor cell lines is facilitated by suppression of OCT4B1.

PURPOSE: The OCT4B1 as a variant of OCT4 is expressed in both cancer cells and tissues. The anti-apoptotic property of this variant aid cancer cells to escape from apoptosis. Therefore, the aim of the present study was to determine the effects of OCT4B1 suppression on regulation of 25 genes involved in anti-apoptotic pathway in tumor cell lines. MATERIAL AND METHODS: AGS (gastric adenocarcinoma), 5637 (bladder tumor) and U-87MG (brain tumor) cells were transfected with specific OCT4B1 siRNA and a scramble siRNA by siRNA silencing gene technology, using Lipofectamine 2000 commercial kit. The real-time PCR technique was employed to examine and calculate fold changes of evaluated genes using the 2-ΔΔCT formula. RESULTS: Present results demonstrated that 22 (88%) of interested genes were similarly down-regulated in all three examined cell lines. Our results also indicated that three genes (CASP2, IGF1R,TNF) were up-regulated. The CFLAR gene was down-regulated in AGS, while it was inversely up-regulated in 5637 and U87MG cells. CONCLUSIONS: It may possibly be concluded that suppression of OCT4B1 can lead to apoptosis in tumor cell lines and this is at least facilitated via down-regulation of examined anti-apoptotic genes. Accordingly, suppression of OCT4B1 may probably be considered as useful tool in cancer therapy and research.

OCT4 spliced variants are highly expressed in brain cancer tissues and inhibition of OCT4B1 causes G2/M arrest in brain cancer cells.

The new claim about the origin of cancer known as Cancer Stem Cell theory states that a somatic differentiated cell can dedifferentiated or reprogrammed for regaining the cancer cell features. It has been recently shown that expression of stemness factors such as Oct4, Sox2, Nanog and Klf4, in a variety of somatic cancers can leads to development of tumorogenesis. Here, the expression of Oct4 variants were evaluated in brain tumor tissues by quantitative RT-PCR and immunohistochemical (IHC) analysis. In next phase of our study, the expression of Oct4B1 was knock-down in brain cancer cell lines and its effect on cell cycle was assessed. Finally, in order to get insights into sequence-structure-function relationships of Oct4 isofroms, their sequences were analysed using bioinformatic tools. Our data revealed that all three variants of Oct4 are expressed in different types of brain cancer. The expression level of Oct4B1, in contast to Oct4B, was much higher in high-grade brain tumors compared with low-grade ones. In line with qPCR, the expression of Oct4A and B isofroms was confirmed with IHC in different types of brain tumors. Moreover, as a result of the suppression of Oct4B1 expression, the brain cancer cells were arrested in G2/M phase of cell cycle. Bioinfromatics data indicated that the predicted Oct4B1 protein have DNA binding properties. All together, our findings suggest that Oct4B1 has a potential role in tumorigenesis of brain cancer and can be considered as a new tumor marker with potential value in diagnosis and treatment of brain cancer.

Oct4B, CD90, and CD73 are upregulated in bladder tissue following electro-resection of the bladder.

AIM: We tested the hypothesis that stimulation by electro-resection of bladder tissue induces stem cells in the tissue repair process. MATERIALS & METHODS: After primary transurethral resection of a bladder tumor and surrounding tissue (TUR-Bt), second TUR-Bt was performed. Tissues excised by second TUR-Bt were immunohistochemically stained for Oct4, a marker of pluripotency, and for CD90 and CD73, markers of mesenchymal stromal cells, when no bladder tumor cells remained. RESULTS AND CONCLUSIONS: Oct4B protein was sporadically stained in the cytoplasm of interstitial cells in four out of eight cases. CD90 and CD73 are upregulated in interstitial and vascular endothelial cells without CD45 expression. Mesenchymal stromal cells, but not pluripotent stem cells, may be mainly involved in bladder tissue repair.

Down-regulation of HSP40 gene family following OCT4B1 suppression in human tumor cell lines.

OBJECTIVES: The OCT4B1, as one of OCT4 variants, is expressed in cancer cell lines and tissues more than other variants and plays an important role in apoptosis and stress (heat shock protein) pathways. The present study was designed to determine the effects of OCT4B1 silencing on expressional profile of HSP40 gene family expression in three different human tumor cell lines. MATERIALS AND METHODS: The OCT4B1 expression was suppressed by specific siRNA transfection in AGS (gastric adenocarcinoma), 5637 (bladder tumor) and U-87MG (brain tumor) cell lines employing Lipofectamine reagent. Real-time PCR array technique was employed for RNA qualification. The fold changes were calculated using RT(2) Profiler PCR array data analysis software version 3.5. RESULTS: Our results indicated that fifteen genes (from 36 studied genes) were down-regulated and two genes (DNAJC11 and DNAJC5B) were up-regulated in all three studied tumor cell lines by approximately more than two folds. The result of other studied genes (19 genes) showed different expressional pattern (up or down-expression) based on tumor cell lines. CONCLUSION: According to the findings of the present study, we may suggest that there is a direct correlation between OCT4B1 expression in tumor cell lines (and tissues) and HSP40 family gene expressions to escape from apoptosis and cancer expansion.

Role of Oct4 in the early embryo development.

Oct4 is a key component of the pluripotency regulatory network, and its reciprocal interaction with Cdx2 has been shown to be a determinant of either the self-renewal of embryonic stem cells (ESCs) or their differentiation into trophoblast. Oct4 of maternal origin is postulated to play critical role in defining totipotency and inducing pluripotency during embryonic development. However, the genetic elimination of maternal Oct4 using a Cre-lox approach in mouse revealed that the establishment of totipotency in maternal Oct4-depleted embryos was not affected, and that these embryos could complete full-term development without any obvious defect. These results indicate that Oct4 is not essential for the initiation of pluripotency, in contrast to its critical role in maintaining pluripotency. This conclusion is further supported by the formation of Oct4-GFP- and Nanog- expressing inner cell masses (ICMs) in embryos with complete inactivation of both maternal and zygotic Oct4 expression and the reprogramming of fibroblasts into fully pluripotent cells by Oct4-deficient oocytes.

Suppression of OCT4B enhances sensitivity of lung adenocarcinoma A549 cells to cisplatin via increased apoptosis.

BACKGROUND: Resistance to chemotherapy in lung adenocarcinoma remains a major obstacle. We examined the potential role of Octamer-binding transcription factor-4B (OCT4B) in enhancing sensitivity of lung adenocarcinoma cells to cisplatin. MATERIALS AND METHODS: RNAi interference was used to examine the role of OCT4B in cisplatin-treated A549 cells. Cells were transfected with OCT4B siRNA prior to a 48-h cisplatin treatment. Propidium iodide (PI) and caspase-3 staining were used to determine cell viability and apoptosis. Cell-cycle analysis was performed to evaluate alterations in phase distribution. RESULTS: OCT4B suppression in cells increased the number of non-viable, PI(+), and apoptotic, caspase-3(+) cells in the presence and absence of cisplatin treatment. Importantly, cisplatin treatment of OCT4B-suppressed cells resulted in a marked transition of cells from G0/G1 to G2/M phase. CONCLUSION: Silencing of OCT4B confers sensitivity to cisplatin treatment in A549 cells via cell-cycle regulation, increased proliferation and enhancement of cisplatin-induced apoptosis. OCT4B clearly protects A549 cells from apoptosis.